Gadd45beta targeting agents

ABSTRACT

Compounds based around tetrapeptide, tripeptide and dipeptide moeties and corresponding peptiod moeties. Related methods and pharmaceutical compositions for use in treatment of cancer, inflammatory diseases, and other disorders.

The work leading to this invention was supported in part by NationalInstitutes of Health R01 Grants CA84040 and CA098583.

FIELD OF INVENTION

The invention relates to cancer and other diseases and disorders forexample inflammatory diseases and disorders and to therapeuticmodulation thereof. In particular, the invention relates to compoundsbased on short peptides capable of modulating programmed cell death(PCD) and proliferation of cancer cells, andpro-inflammatory/auto-immune cells.

BACKGROUND OF THE INVENTION

The induction of apoptosis has long been considered as a method oftargeting cancer cells as well as pro-inflammatory, autoimmune cells,and other diseased cells. There are a number of cellular pathwaysinvolved in triggering cell death including the c-Jun N-terminal kinaseJNK pathway. JNKs are responsive to cytokines and stress stimuli such asultraviolet irradiation, heat shock and osmotic shock. Also activated inthe response to cytokines and cellular stress is the NF-κB pathway. TheNF-κB pathway can inhibit the JNK pathway by crosstalk mediated byGadd45β and the JNK kinase, mitogen activated protein-kinase kinase 7(MKK7/JNKK2). MKK7 activity is inhibited by Gadd45β, a member of theGadd45 family of inducible factors and a direct transcriptional targetof NF-κB. This means that Gadd45β mediates NF-κB suppression of JNKsignalling by binding to MKK7 and inhibiting its activity. Papa, et al.2004, Nature Cell Biology 6(2):1462153.

The use of NF-κB inhibitors has been proposed for use in the treatmentof cancer and inflammatory diseases. However, because NF-κB has a numberof activities including roles in PCD, immunity, inflammation and tissuedevelopment, it is preferred to inhibit specific functions of NF-κBrather than NF-κB itself.

The present invention relates to the inhibition of Gadd45β which isknown to be up-regulated in a number of cancers and also in chronicinflammatory and hereditary disorders.

Multiple myeloma (MM), also known as plasma cell myeloma or Kahler'sdisease, is a cancer of plasma cells. Multiple myeloma is currentlyincurable, although temporary remissions can be induced by use ofsteroids, chemotherapy, thalidomide, proteasome inhibitors (PIs), e.g.bortezomib, melphalan, and stem cell transplants. According to theAmerican Cancer Society, there are approximately 45,000 people in theUnited States living with multiple myeloma with approximately 15,000 newcases being diagnosed each year in the United States. The averagesurvival time from diagnosis is approximately three years. Multiplemyeloma is the second most prevalent blood cancer after non-Hodgkin'slymphoma and represents approximately 1% of all cancers andapproximately 2% of all cancer deaths. The incidence of multiple myelomaappears to be increasing and there is also some evidence that the age ofonset of the disease is falling. Thus, there is a clear need forimproved treatments for multiple myeloma.

Nearly all multiple myeloma primary tumours and multiple myeloma celllines display constitutive NF-κB activity. Blocking the activity ofNF-κB causes multiple myeloma cell death. A major barrier to achievinglong-term cancer treatment results with NF-κB targeting strategies islack of specificity, and therefore poor treatment tolerability. This isdue to the pleiotropic functions of NF-κB and of the proteasome. Thereis a need for a radically new therapeutic approach which is morespecific, safer, and therefore more effective.

One of NF-κB's key functions in multiple myeloma is to promote survival.It has been shown (De Smaele, et al. (2001) Nature 414:306-313) thatNF-κB affords cyto-protection by suppressing the JNK MAPK cascade bymeans of Gadd45β, a member of the Gadd45 family of inducible factors.Gadd45β is up-regulated by NF-κB in response to various stimuli andpromotes survival by directly targeting the JNK kinase MKK7 (Papa, etal. 2004 Nature Cell Biology 6:146-153, Papa, et al. 2007) J. Biol.Chem. 282:19029-19041, Papa, et al. (2008) J. Clin. Invest.118:191-1923).

Proteasome inhibitors (PIs) and direct NF-κB inhibitors kill multiplemyeloma cells by activating the JNK pathway, but are unsuitable forcurative multiple myeloma therapy because of their indiscriminateeffects on NF-κB and/or indiscriminate effects on the proteasome whichprevents them being used at fully inhibitory curative doses.

In addition to multiple myeloma, Gadd45β is expressed at high levels inother tumours including diffuse large B-cell lymphoma, Burkitt'slymphoma, promonocytic leukaemia and other leukemias, as well as somesolid tumours including hepatocellular carcinoma, bladder cancer, brainand central nervous system cancer, breast cancer, head and neck cancer,lung cancer, and prostate cancer. Therefore, inhibiting Gadd45β in thesetumours may induce cancer cell death and so have beneficial therapeuticeffects. Many haematological malignancies (including multiple myeloma,mantle cell lymphoma, MALT lymphoma, diffuse large B-cell lymphoma,Hodgkin's lymphoma, myelodysplastic syndrome, adult T-cell leukaemia(HTLV-1), chronic lymphocytic leukaemia, chronic myeloid leukaemia,acute myelogenic leukaemia, and acute lymphocytic leukaemia) and solidtumours (including breast cancer, cervical cancer, renal cancer, lungcancer, colon cancer, liver cancer, oesophageal cancer, gastric cancer,laryngeal cancer, thyroid cancer, parathyroid cancer, bladder cancer,ovarian cancer, prostate cancer, pancreatic cancer and many othercancers) are also known to exhibit constitutive NF-κB activationproviding pro-survival signals to the cells at the expense of PCD whichcould otherwise lead to increased tumour cell death (V. Baud and M.Karin 2009, Nat. Rev. Drug Disc. 8: 33-40). Constitutive NF-κB activityis also found in melanoma, cylindroma, squamous cell carcinoma (skin,and head and neck), oral carcinoma, endometrial carcinoma,retinoblastoma, astrocytoma, and glioblastoma (V. Baud and M. Karin2009, Nat. Rev. Drug Disc. 8: 33-40). Inhibiting Gadd45β in thesetumours featuring aberrantly high constitutive NF-κB activity could alsoproduce beneficial therapeutic effects by inducing programmed cell deathin the cancerous cells. The present invention is based on therealisation that targeting the discreet pro-survival functions of NF-κBin cell survival via Gadd45β provides safer, more effective, therapythan does targeting NF-κB directly for a range of diseases and disordersincluding cancer and also other diseases characterised by aberrant cellsurvival or diseases which could be treated by the induction ofincreased PCD (such as autoimmune diseases, chronic inflammatorydiseases, degenerative diseases and ischemic and vascular diseases).

A broad range of diseases and disorders depend on the activity of NF-κB.Indeed, the pathogenesis of virtually every known human disease ordisorder is now being considered to depend on inflammation, and hence toinvolve NF-κB. This functions as a masterswitch of the inflammatoryresponse, coordinating expression of an array of over 200 genes encodingcytokines, receptors, transcription factors, chemokines,pro-inflammatory enzymes, and other factors, including pro-survivalfactors, which initiate and sustain inflammation. The compounds of theinvention inhibit the discrete pro-survival activity of NF-κB ininflammation. Therefore, diseases and disorders amenable to treatmentwith these compounds include, apart from conventional chronicinflammatory diseases (such as inflammatory bowel disease, rheumatoidarthritis, and psoriasis), other diseases and disorders that depend on asignificant inflammatory component. Examples of such diseases anddisorders, which are being treated with anti-inflammatory agents orNF-κB-inhibiting agents or have been proposed as suitable for treatmentwith NF-κB inhibitors and could also be treated with a compound of theinvention, include:

1. respiratory tract: obstructive diseases of the airways including:asthma, including bronchial, allergic, intrinsic, extrinsic,exercise-induced, drug-induced (including aspirin and NSAID-induced) anddust-induced asthma, both intermittent and persistent and of allseverities, and other causes of airway hyper-responsiveness; chronicobstructive pulmonary disease (COPD); bronchitis, including infectiousand eosinophilic bronchitis; emphysema; bronchiectasis; cystic fibrosis;sarcoidosis; farmer's lung and related diseases; hypersensitivitypneumonitis; lung fibrosis, including cryptogenic fibrosing alveolitis,idiopathic interstitial pneumonias, fibrosis complicatinganti-neoplastic therapy and chronic infection, including tuberculosisand aspergillosis and other fungal infections; complications of lungtransplantation; vasculitic and thrombotic disorders of the lungvasculature, and pulmonary hypertension; antitussive activity includingtreatment of chronic cough associated with inflammatory and secretoryconditions of the airways, and iatrogenic cough; acute and chronicrhinitis including rhinitis medicamentosa, and vasomotor rhinitis;perennial and seasonal allergic rhinitis including rhinitis nervosa (hayfever); nasal polyposis; acute viral infection including the commoncold, and infection due to respiratory syncytial virus, influenza,coronavirus (including SARS) or adenovirus; or eosinophilic esophagitis;2. bone and joints: arthritides associated with or includingosteoarthritis/osteoarthrosis, both primary and secondary to, forexample, congenital hip dysplasia; cervical and lumbar spondylitis, andlow back and neck pain; osteoporosis; rheumatoid arthritis and Still'sdisease; seronegative spondyloarthropathies including ankylosingspondylitis, psoriatic arthritis, reactive arthritis andundifferentiated spondarthropathy; septic arthritis and otherinfection-related arthopathies and bone disorders such as tuberculosis,including Potts' disease and Poncet's syndrome; acute and chroniccrystal-induced synovitis including urate gout, calcium pyrophosphatedeposition disease, and calcium apatite related tendon, bursal andsynovial inflammation; Behcet's disease; primary and secondary Sjogren'ssyndrome; systemic sclerosis and limited scleroderma; systemic lupuserythematosus, mixed connective tissue disease, and undifferentiatedconnective tissue disease; inflammatory myopathies includingdermatomyositits and polymyositis; polymalgia rheumatica; juvenilearthritis including idiopathic inflammatory arthritides of whateverjoint distribution and associated syndromes, and rheumatic fever and itssystemic complications; vasculitides including giant cell arteritis,Takayasu's arteritis, Churg-Strauss syndrome, polyarteritis nodosa,microscopic polyarteritis, and vasculitides associated with viralinfection, hypersensitivity reactions, cryoglobulins, and paraproteins;low back pain; Familial Mediterranean fever, Muckle-Wells syndrome, andFamilial Hibernian Fever, Kikuchi disease; drug-induced arthalgias,tendonititides, and myopathies;3. pain and connective tissue remodelling of musculoskeletal disordersdue to injury [for example sports injury] or disease: arthitides (forexample rheumatoid arthritis, osteoarthritis, gout or crystalarthropathy), other joint disease (such as intervertebral discdegeneration or temporomandibular joint degeneration), bone remodellingdisease (such as osteoporosis, Paget's disease or osteonecrosis),polychondritits, scleroderma, mixed connective tissue disorder,spondyloarthropathies or periodontal disease (such as periodontitis);4. skin: psoriasis, atopic dermatitis, contact dermatitis or othereczematous dermatoses, and delayed-type hypersensitivity reactions;phyto- and photodermatitis; seborrhoeic dermatitis, dermatitisherpetiformis, lichen planus, lichen sclerosus et atrophica, pyodermagangrenosum, skin sarcoid, discoid lupus erythematosus, pemphigus,pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitides,toxic erythemas, cutaneous eosinophilias, alopecia greata, male-patternbaldness, Sweet's syndrome, Weber-Christian syndrome, erythemamultiforme; cellulitis, both infective and non-infective; panniculitis;cutaneous lymphomas, non-melanoma skin cancer and other dysplasticlesions; drug-induced disorders including fixed drug eruptions;5. eyes: blepharitis; conjunctivitis, including perennial and vernalallergic conjunctivitis; iritis; anterior and posterior uveitis;choroiditis; autoimmune; degenerative or inflammatory disordersaffecting the retina; ophthalmitis including sympathetic ophthalmitis;sarcoidosis; infections including viral, fungal, and bacterial;6. gastrointestinal tract: glossitis, gingivitis, periodontitis;oesophagitis, including reflux; eosinophilic gastro-enteritis,mastocytosis, Crohn's disease, colitis including ulcerative colitis,proctitis, pruritis ani; coeliac disease, irritable bowel syndrome, andfood-related allergies which may have effects remote from the gut (forexample migraine, rhinitis or eczema);7. abdominal: hepatitis, including autoimmune, alcoholic and viral;fibrosis and cirrhosis of the liver; cholecystitis; pancreatitis, bothacute and chronic;8. genitourinary: nephritis including interstitial andglomerulonephritis; nephrotic syndrome; cystitis including acute andchronic (interstitial) cystitis and Hunner's ulcer; acute and chronicurethritis, prostatitis, epididymitis, oophoritis and salpingitis;vulvo-vaginitis; Peyronie's disease; erectile dysfunction (both male andfemale);9. allograft rejection: acute and chronic following, for example,transplantation of kidney, heart, liver, lung, bone marrow, skin orcornea or following blood transfusion; or chronic graft versus hostdisease;10. CNS: Atzheimer's disease and other dementing disorders including CJDand nvCJD; amyloidosis; multiple sclerosis and other demyelinatingsyndromes; cerebral atherosclerosis and vasculitis; temporal arteritis;myasthenia gravis; acute and chronic pain (acute, intermittent orpersistent, whether of central or peripheral origin) including visceralpain, headache, migraine, trigeminal neuralgia, atypical facial pain,joint and bone pain, pain arising from cancer and tumor invasion,neuropathic pain syndromes including diabetic, post-herpetic, andHIV-associated neuropathies; neurosarcoidosis; central and peripheralnervous system complications of malignant, infectious or autoimmuneprocesses;11. other auto-immune and allergic disorders including Hashimoto'sthyroiditis, Graves' disease, Addison's disease, diabetes mellitus,idiopathic thrombocytopaenic purpura, eosinophilic fasciitis, hyper-IgEsyndrome, antiphospholipid syndrome;12. other disorders with an inflammatory or immunological component;including acquired immune deficiency syndrome (AIDS), leprosy, Sezarysyndrome, and paraneoplastic syndromes;13. cardiovascular: atherosclerosis, affecting the coronary andperipheral circulation; pericarditis; myocarditis, inflammatory andauto-immune cardiomyopathies including myocardial sarcoid; ischaemicreperfusion injuries; endocarditis, valvulitis, and aortitis includinginfective (for example syphilitic); vasculitides; disorders of theproximal and peripheral veins including phlebitis and thrombosis,including deep vein thrombosis and complications of varicose veins;14. gastrointestinal tract: Coeliac disease, proctitis, eosinopilicgastro-enteritis, mastocytosis, Crohn's disease, ulcerative colitis,microscopic colitis, indeterminant colitis, irritable bowel disorder,irritable bowel syndrome, non-inflammatory diarrhea, food-relatedallergies which have effects remote from the gut, e.g., migraine,rhinitis and eczema.

The present invention relates to novel inhibitors of the Gadd45β/MKK7complex and/or signalling of that complex which may be used to inhibitthe pro-survival function of NF-κB in cancer, inflammation, autoimmunityand degenerative, ischemic and vascular disorders.

SUMMARY OF INVENTION

According to a first aspect of the invention there is provided acompound of formula I:X₁-A-X₂  I:

-   -   wherein,    -   A is A″″,        -   or A″-[M-A′-]_(n) M-A′″;    -   A″ is A″,        -   A′″        -   or Z₁—Y₂—Y₃—Z₄, wherein Y₂—Y₃ is an oligopeptide moiety or            an oligopeptoid moiety having the residues Y₂—Y₃ and Z₁ is            attached to the N-terminal nitrogen of Y₂—Y₃ and Z₄ is            attached to the C-terminal carbon of Y₂—Y₃;    -   A″ is A′,        -   or Y₁—Y₂—Y₃—Z₄, wherein Y₁—Y₂—Y₃ is an oligopeptoid moiety            or an oligopeptoid moiety comprising the residues: Y₁—Y₂—Y₃            and Z₄ is attached to the C-terminal carbon of Y₁—Y₂—Y₃;    -   A′″ is A′,        -   or Z₁—Y₂—Y₃—Y₄, wherein Y₂—Y₃—Y₄ is an oligopeptoid moiety            or an oligopeptoid moiety comprising the residues Y₂—Y₃—Y₄            and Z₁ is attached to the N-terminal nitrogen of Y₂—Y₃—Y₄;    -   each occurrence of A′ is independently an oligopeptide moiety or        an oligopeptoid moiety comprising the residues Y₁—Y₂—Y₃—Y₄;    -   n is an integer from 0 to 18    -   Y₁ and Y₄ are independently amino acid residues or residues of        amino acid derivatives having aromatic side chains    -   Y₂ is an amino acid residue or a residue of an amino acid        derivative or is absent,    -   Y₃ is an amino acid residue or a residue of an amino acid        derivative or is absent;        Z₁ is a group of formula II:

which is linked to the N-terminal nitrogen of Y₂,W is absent, or an oxygen, or a nitrogen, or an alkylene group of fromone to three carbons,which alkylene group of from one to three carbons is optionallysubstituted by at least one substituent selected from alkyl of from oneto four carbons, or 5-10 membered carbocyclic or heterocyclic aromaticgroup;J is a 5-10 membered carbocyclic or heterocyclic aromatic group,which aromatic group is optionally substituted by at least onesubstituent selected from hydroxyl, halogen, alkyl of from one to fourcarbons, or alkoxy of from one to four carbon atoms;Z₄ represents a group of formula III:

which is linked to the C-terminal carbon of Y₃,R is hydrogen or alkyl of from one to four carbons;W′ is absent or an alkylene group of from one to three carbons, whichalkylene group of from one to three carbons is optionally substituted byat least one substituent selected from alkyl of from one to fourcarbons, or 5-10 membered carbocyclic or heterocyclic aromatic group;J′ is a 3-10 membered aliphatic carbocyclic group or a 5-10 memberedcarbocyclic or heterocyclic aromatic group, which aliphatic or aromaticgroup is optionally substituted by at least one substituent selectedfrom hydroxyl, halogen, alkyl of from one to four carbons, or alkoxy offrom one to four carbon atoms;M is a peptide bond between preceding oligopeptide or oligopeptoidmoiety (A′, A″ or A′″) and following oligopeptide or oligopeptide moiety(A′, A″ or A′″) or a linker moiety attached via an amide bond, an esterbond, an ether bond, or a thioether bond to the terminal carboxylicgroup of preceding oligopeptide or oligopeptoid moiety (A′, A″ or A′″)and via an amide bond, an ester bond, an ether bond, or a thioether bondto the terminal amino group of following oligopeptoid moiety (A′, A″ orA′″);X₁ is absent, or is a moiety added to the -amino terminal of A in orderto block the free amino group;X₂ is absent or is a moiety added to the carboxyl terminal of A in orderto block the free carboxyl group;with the proviso that X₁ is absent if A comprises Z₁ and X₂ is absent ifA comprises Z₄;or derivatives thereof, said derivatives being selected from the groupconsisting of:

-   -   a) oligomers or multimers of molecules of the compound of        formula I, said oligomers and multimers comprising two or more        molecules of the compound of formula I each linked to a common        scaffold moiety via an amide bond formed between an amino or        carboxylic acid group present in molecules of the compound of        formula I and an opposite amino or carboxylic acid group on a        scaffold moiety said scaffold moiety participating in at least 2        amide bonds,    -   b) derivatives comprising a molecule of the compound of formula        I or an oligomer or multimer thereof as defined above in part a)        conjugated via an amide bond, an ester bond, an ether bond or a        thioether bond to:        -   PEG,        -   PEG-based compounds,        -   cell-penetrating peptides,        -   fluorescent dyes,        -   biotin or other tag moiety,        -   fatty acids,        -   nanoparticles of discrete size        -   or chelating ligands complexed with metallic or        -   radioactive ions.    -   c) derivatives comprising a molecule of the compound of formula        I or an oligomer or multimer thereof as defined in part a) which        has been modified by amidation, glycosylation, carbamylation,        acylation, sulfation, phosphorylation, cyclization, lipidation,        pegylation or linkage to a peptide or peptiod fusion partner to        make a fusion peptide or fusion peptiod.        and    -   d) salts and solvates of a molecule of the compound of formula I        or of a derivative thereof as defined in part a) or b) above.

According to a second aspect of the invention, there is provided apharmaceutical composition comprising a compound according the firstaspect of the invention and a pharmaceutically acceptable carrier.

According to a third aspect of the invention, there is provided a methodof treating a disease or disorder characterised by increased NF-κBactivity and/or expression and/or increased Gadd45β activity and/orexpression comprising administering a therapeutically effective amountof a compound according to the first aspect of the invention or apharmaceutical composition according to the second aspect of theinvention to a subject in need thereof.

According to a fourth aspect of the invention, there is provided acompound according to the first aspect of the invention or a compositionaccording to the second aspect of the invention for use as a medicament.

According to a fifth aspect of the invention, there is provided use of acompound according to the first aspect of the invention or apharmaceutical composition according to the second aspect of theinvention for the manufacture of a medicament for the treatment of adisease or disorder characterised by increased NF-κB activity and/orexpression and/or increased Gadd45β activity and/or expression.

BRIEF EXPLANATION OF DRAWINGS

FIG. 1. Schematic representation of the protective crosstalk between theNF-κB and JNK pathways in the context of TNF-R1 signalling. It can beseen that Gadd45β mediates crosstalk between the survival pathwayinduced by NF-κB and the death pathway induced by MKK7 and JNK.Inhibition of this crosstalk by blocking Gadd45β allows MKK7 to activateJNK thus triggering a death pathway in tumour cells.

FIG. 2. Model of the Gadd45β-MKK7 complex. The model was built asreported in the reference by Papa S. et al. 2007, J Biol Chem 282:19029-19041. The model was further refined using the crystallographicstructure of MKK7 (pdb: 2DYL) and a structure of Gadd45β modelled on thecrystallographic structure of Gadd45γ (pdb: 3FFM).

FIG. 3. (A) The ELISA screen used to isolate the lead D-tetrapeptides(DTPs) 1 and 2. Antagonists of the Gadd45β/MKK7 interaction wereselected by screening a simplified combinatorial peptide library ofgeneral formula Fmoc-(βAla)₂-X₁—X₂—X₃—X₄—CONH₂ (see reference by Marascoet al. 2008, Curr. Protein Pept. Sci. 9:447-67) prepared using one of 12amino acids at each position from X₁ to X₄. This library, containing atotal of 12⁴=20,736 different peptides, was iteratively deconvoluted infour steps by ELISA competition assays, using at each step coated humanMKK7 (42 nM), soluble biotin-labeled human (h)Gadd45β (21 nM) and eachof the 12 sub-libraries at the nominal concentration of 42 nM (notshown). (B) The most active peptide of first generation was then usedfor the synthesis of a second-generation library. The screening of thislibrary provided two highly active peptides (labelled in FIG. 3B as 1and 8). (C) Optimized peptides were then freed of the Fmoc-(βAla)₂-tagand synthesized as D-isomers, yielding DTP1 and DTP2, which disruptedthe Gadd45β/MKK7 interaction with IC₅₀ of 0.22 nM and 0.19 nM,respectively. It can also be seen that the L-isomers of these peptides(i.e. LTP1 and LTP2) exhibited IC₅₀s similar to those of DTPs in theELISA competition assays, whereas the negative control peptides, LNC andDNC, displayed no detectable inhibitory effect on the formation of theGadd45β/MKK7 complex. LNC, L-isomer negative control; LTP1, L-isomertetrapeptide 1; LTP2, L-isomer tetrapeptide 2; DNC, D-isomer negativecontrol.

FIG. 4. Stability of Z-DTPs in biological fluids. ELISA competitionassays showing that the Z-protected DTPs (Z-DTP1, Z-DTP2) retain fullinhibitory activity after a 48-hr incubation with human serum at 37° C.(IC₅₀=0.19 nM, Z-DTP1; IC₅₀=0.18 nM, Z-DTP2), whereas the Z-protectedLTPs (Z-LTP1, Z-LTP2) are almost completely inactivated after thistreatment (IC₅₀s>10 μM). Assays were performed as in FIG. 3C, usingcoated MKK7, soluble biotin-hGadd45β, and the indicated concentrationsof the tetrapeptides. Z-LNC and Z-DNC, L- and D-isomer negativecontrols, respectively.

FIG. 5. Co-immunoprecipitation (co-IP) assays showing the effective andspecific disruption of the Gadd45β/MMK7 interaction by D-tetrapeptides 1and 2 (DTP1 and DTP2), but not by negative control (NC) D-tetrapeptides(NC1, NC2, NC3 and NC4). Co-IP was performed using anti-FLAG (MKK7)antibody, and western blots were developed using anti-HA (detectingHA-Gadd45β) (top) or anti-MKK7 (bottom) antibodies, as indicated.

FIG. 6. MKK7 kinase assays showing the effective and specific disruptionof the Gadd45β/MMK7 interaction and the restoration of MKK7 catalyticactivity by D-tetrapeptides 1 and 2 (DTP1 and DTP2), but not by negativecontrol (NC) D-tetrapeptides (NC1, NC2, NC3 and NC4). Active MKK7 wasimmunoprecipitated with anti-FLAG antibody from phorbol 12-myristate13-acetate (PMA)/ionomycin (P/I)-treated HEK-293T cells and incubatedwith the D-tetrapeptides in the presence (top panel) or absence (bottompanel) of recombinant human (h)Gadd45β, as indicated. As shown, neitheractive lead D-tetrapeptides nor control NC tetrapeptides inhibited MKK7catalytic activity when incubated with the kinase in the absence ofGadd4513 (bottom panel).

FIG. 7. (A, B, C) [³H]thymidine incorporation assays, showing thatZ-protected derivatives of DTP2 (Z-DTP2), but not the acetyl derivativesof DTP2 (Ac-DTP2) or the Z-protected derivatives of the L-isomers ofDTP2 (Z-LTP2) have significant tumoricidal activity in tumour celllines. Data are expressed as percentage of survival/proliferation oftumour cells after treatment with either 10 μM of Z-DTP2 (A), Ac-DTP2(B) or Z-LTP2 (C) (filled columns), or with Z-DNC (A), Ac-DNC (B) andZ-LNC (C) (empty columns) relative to the survival/proliferation ofuntreated cells. Time points are indicated. Shown are 3 out of the 8susceptible multiple myeloma cell lines tested (i.e. U266, KMS-11,NCI-H929), and the Burkitt's lymphoma (BJAB) and pro-monocytic leukaemia(U937) cell lines. These data establish the high cytotoxic activity ofZ-DTP2 (A) compared to the inactivity of Ac-DTP2 (B) and the lowactivity of Z-LTP2 (C) (see also FIGS. 8A, 8B, and 8C and Table IV;additional multiple myeloma lines). (B) The absence of Ac-DTP2'stumoricidal activity in multiple myeloma cell lines correlated with thelow cellular permeability of this compound, as established in CaCO2assays (data not shown). The viability of multiple myeloma cell linesafter treatment with other, less effective DTPs' derivatives (alsodesigned to improve DTPs' cellular uptake), including those bearing amethyl (Me), acetyl (Ac), myristyl (Myr), 3-methoxy, 4-hydroxy-benzoyl,benzoyl, 6Cl-benzyloxycarbonyl (6Cl-Z), and/orfluorenylmethyloxycarbonyl (Fmoc) group, is not shown. (C) AlthoughZ-LTPs' in vitro potency and cellular uptake were comparable to those ofZ-DTPs (see FIG. 3C; also data not shown), Z-LTP2 shows low activity inmultiple myeloma cells, due to low stability in biological fluids (seeFIG. 4).

FIG. 8. Z-DTPs' proapoptotic activity is selective for tumour cell lineswith constitutive NF-κB activity. (A, B, C) [³H]Thymidine incorporationassays, performed as described in FIG. 7, showing cell survival in apanel of tumour cell lines after treatment with 10 μM Z-DTPs or Z-DNCfor the following times: 144 hrs (A); 24 hrs, 72 hrs or 144 hrs (B, C),as indicated. (A) Shown is the potent tumoricidal activity of Z-DTP2 in8 out of 9 multiple myeloma cell lines, 1 out of 2 diffuse large B-celllymphoma (DLBCL; LY3) cell lines, 1 out of 1 promonocytic leukemia cellline (U937), and in 1 out of 6 Burkitt's lymphoma cell lines (BJAB) thatwere tested (see also FIG. 9). Interestingly, Z-DTP2 showed cytotoxicactivity only in the DLBCL cell line of the activated-B-cell (ABC)-likesubtype (i.e. LY3), and not in that of the germinal center B-cell(GCB)-like (i.e. SUDHL6) subtype, which does not feature constitutiveNF-κB activation (Ngo V N, et al. Nature 441(7089):106-10; see also FIG.12, levels of Gadd45β expression). They also show activity in multiplemyeloma cells lines, virtually all of which feature constitutive NF-κBactivation. Tumoricidal activity of Z-DTP2 (B) and Z-DTP1 (C) inmultiple myeloma and DLBCL cell lines after treatment with 10 μM ofZ-DTP1, Z-DTP2 and Z-DNC for the times indicated (i.e. 24, 72 or 144hrs, as shown). Results were confirmed in trypan blue exclusion assays(data not shown) and propidium iodide (PI) assays (see FIG. 10; alsodata not shown).

FIG. 9. [³H]Thymidine incorporation assays showing absence of Z-DTP2cytotoxicity in a panel of 22 resistant tumour cell lines aftertreatment with Z-DTP2 for 144 hours, even when this compound was used atvery high concentrations—that is 100 μM. Z-DNC, Z-protected D-negativecontrol. Also shown are the sensitive cell lines BJAB (Burkitt'slymphoma), KMS-11 and KMS-12 (multiple myeloma). Notably, there was astrong correlation in these cell lines between sensitivity toZ-DTP2-induced killing and levels of endogenous Gadd45β expression (seeFIGS. 12A and 12B).

FIGS. 10A, 10B, 10C, 10D and 10E. Z-DTP2-induced killing in multiplemyeloma cell lines is due to apoptosis. Propidium iodide (PI) nuclearstaining assays showing the induction of apoptosis (i.e. sub-G₁ DNAcontent; see FL2-A) in the representative multiple myeloma cell lines,NCI-H929 (Figure A) KMS-11, (Figure B), ARH-77, (Figure C) JJN-3,(Figure D) and U266, (Figure E) after treatment with 10 μM of Z-DTP2 orZ-DNC1, as shown, for 72 or 144 hrs. Also shown is the DNA content ofuntreated cells cultured under the same conditions. Percentages ofapoptotic cells are depicted in the histograms.

FIG. 11. Z-DTP2 treatment causes strong JNK activation in multiplemyeloma cell lines. KMS11 and NCI-H929 cells were treated with 10 μM ofZ-DTP2 or Z-DNC, as shown, and JNK activation was monitored at theindicated times by western blotting using an anti-phospho(P)-JNK-specific antibody. Increased JNK phosphorylation (a marker ofJNK activation) is only seen after treatment with Z-DTP2, but not aftertreatment with Z-protected negative control peptide (Z-DNC). TNFαstimulation (2,000 U/ml) was used as positive control for JNKactivation. Importantly, similar effects of Z-DTP2 were seen on MKK7activation (data not shown). Moreover, as seen with the biologicalactivity of Gadd45β (see references: De Smaele, et al. (2001) Nature414:306-313; Papa, S et al., (2004) Nat. Cell Biol. 6, 146-153; Papa, etal. 2007 J. Biol. Chem. 282:19029-19041; Papa, et al. (2008) J. Clin.Invest. 118:191-1923), the effects of Z-DTPs in multiple myeloma celllines were specific for the MKK7/JNK pathway, as no effects wereobserved with these compounds on the activation of the IKK/NF-κB, ERKand p38 pathways in these cell lines (data not shown).

FIG. 12. Strong correlation in tumour cell lines between cellsensitivity to Z-DTP-induced killing and levels of Gadd45β expression.(A) The top panel shows the expression of Gadd45β in a panel of 29cancer cell lines (qRT-PCR; red columns); whereas the bottom panel showsthe percentage of cell death in the same cell lines after treatment with10 μM of Z-DTP2 for 144 hrs ([³H]thymidine incorporation; blackcolumns). (B) Shown is the correlation plot of Gadd45β expression versusthe percentage of cell survival after treatment with Z-DTP2 for the sameexperiment shown in (A). The significance of the correlation coefficientbetween the 2 parameters' domain is very high (p<0.01) (Pearsoncorrelation, which quantifies the association between two variables,calculated using the GraphPad software). These data confirm the hightarget specificity of Z-DTPs in cells. Values in (A) (top panel) werenormalized to β-actin.

FIG. 13. Chemical structures of relevant compounds disclosed in thispatent and description of possible pharmacophores and strategies fortheir assessment. (A) Shown are the chemical structures of the parentcompound Z-DTP2 (Z-D-Tyr-D-Glu-D-Arg-D-Phe-NH₂) [SEQ ID NO.: 1] and ofthe Z-DTP2 derivatives, mDTP1(p-hydroxy-benzoic-acid-D-Glu-D-Arg-phenetylamine), mDTP2(Ac-D-Tyr-D-Glu-D-Phe-NH₂), and mDTP3 (Ac-D-Tyr-D-Arg-D-Phe-NH₂). Thesemodified Z-DTP2 compounds (hereafter termed mDTPs) were tested foractivity both in vitro (ELISA) and in cells (killing assays). Themolecular weights (MW), IC₅₀s in vitro and in cells and ligandefficiency of Z-DPT2 and of these representative modified compounds arealso reported (see also Table V). (B) Outlined are the main steps of thestrategy achieved to identify the possible pharmacophore of thebioactive compounds (Geeson M P. 2008 J Med Chem. 51:817-834). Most ofthe proposed changes have already been explored: N-terminal groups (seeTable III); Tyr to cyclohexylalanine, Phe to cyclohexylalanine exchange,removal of the internal Glu and/or Arg, exchange of Glu to Asp, esterprodrugs on Asp side chain (see Table V); Tyr to Phe swap, exchange Argto His, Lys or Pro (see Table VI). Together, the data show that thebioactive pharmacophore can be described as follows: a tyrosine or asimilar aromatic ring with H-bond donor/acceptors needed on position Y₁;at least one alpha-amino acid needed on position Y₂ and/or Y₃,preferably with a basic group to improve cellular uptake. Proline,asparagines, or leucine at position Y₂ with or without arginine onposition Y₃ also allow the retention of bioactivity. A distance greaterthan about 7 Angstrom between the two aromatic rings (i.e. a distancegreater than that imposed by one alpha-amino acid) causes a reduction inbioactivity; an aromatic ring is needed at position Y₄, with or withoutH-bond donor/acceptor groups for retention of bioactivity (Table VI).

FIG. 14. (A, B, C, D, E) Cytotoxic activity of Z-DTPs in primarymultiple myeloma cells isolated from 5 representative patients. Eachpanel depicts the data obtained with cells from a different patient—thatis patient 1 (A), patient 2 (B), patient 3 (C), patient 4 (D), andpatient 5 (E). (A, B, C, D, E) Treatments with Z-DTP2, Z-DTP1 and Z-DNCwere at the concentrations indicated, for 48 hrs. Also shown are theuntreated cells from each patient (−). Assays were performed usingtrypan blue exclusion and cell counting. Values represent the percentageof live cells observed after treatment with Z-DTP2, Z-DTP1 or Z-DNCrelative to the viability of untreated control cells.

FIG. 15. Absence of cytotoxic activity of Z-DTPs in primaryuntransformed cells from multiple myeloma-free individuals, includingbone marrow stromal cells (BMSCs) (A), peripheral blood mononuclearcells (PBMNCs) (A), and mesenkymal stem cells (MSCs) (B), or in purifiedprimary B- and T-lymphocytes from mice (B). Treatments with Z-DTP2,Z-DTP1 and Z-DNC were at the concentrations indicated, for either: 48hrs (BMSCs, PBMNCs) (A), 72 hrs (murine B and T cells) (B), or 144 hrsMSCs (B). Assays were performed using trypan blue exclusion and cellcounting (A) or [³H]thymidine incorporation (B).

FIG. 16. Induction of cell death in representative multiple myeloma celllines after sh-RNA-mediated silencing of Gadd45β expression. (A, B, C)The Z-DTP-sensitive multiple myelom cell lines ARH-77 (A) and NCI-H929(B) and the Z-DTP-resistant multiple myeloma cell line, RPMI-8226 (C),were infected with lentivirus-expressing either Gadd45β-specific sh-RNAs(i.e sh-Gadd45β-1, sh-Gadd45β-2, or sh-Gadd45β-3), MKK7-specific sh-RNAs(i.e. sh-MKK7-1 or sh-MKK7-2), or non-specific sh-RNAs (i.e. sh-NS-1 orsh-NS-2), and the viability of infected cells was monitored over aperiod of 8 days by using flow cytometry—revealing cells expressingenhanced green fluorescent protein (eGFP), that is infected cells—andcell counting. Shown is the percent survival of eGFP⁺ (that is infected)multiple myeloma cells at the times indicated relative to the viabilityof eGFP⁺ multiple myeloma cells in the same culture at day 0. (A, B, C)Cells were infected with pLentiLox.3.7 lentiviruses expressing theindicated sh-RNAs as well as eGFP, using standard methods (as reportedin the reference by Yang H et al., Proc Natl Acad Sci USA. 2006 Jul. 5;103(27):10397-402). 5 days later, eGFP⁺ cells were sorted using a BDFACSAria™ II cell sorter, then left to rest for 2 days before beginningthe analyses of cell viability. This time (that is the start of theviability analyses) is denoted in the graphs as day 0. The data showthat the inhibition of Gadd45β expression causes rapid cell death inmultiple myeloma cell lines that are sensitive to Z-DTP-induced toxicity(that is the ARH-77, NCI-H929 cell lines) (A, B), but not in theRPMI-8226 multiple myeloma cell line (C), which is resistant to thistoxicity. These data further establish the target specificity of Z-DTPsfor the Gadd45β/MKK7 complex in multiple myeloma cells (see also FIGS.7, 8, 9, and 12; killing and qRT-PCR assays). They also demonstrate theessential role that Gadd45β plays in multiple myeloma cell survival,thus further validating Gadd45β as a therapeutic target in multiplemyeloma.

FIG. 17. (A, B) [³H]Thymidine incorporation assays showing that thesh-RNA-mediated silencing of Gadd45β, but not that of MKK7, has potenttumouricidal activity in multiple myeloma cell lines that aresusceptible to Z-DTPs-induced killing (that is the ARH-77 and NCI-H929cell lines; see also FIGS. 7A, 7B, 7C and 8, sensitivity toZ-DTP-induced killing). Viability of the Z-DTP-resistant multiplemyeloma cell line, RPMI-8226, is instead unaffected by sh-RNA-mediatedGadd45β inhibition. (A) Shown is the viability of the threerepresentative multiple myeloma cell lines, RPMI-8226, NCI-H929 andARH-77, after the silencing of Gadd45β or MKK7. (B) Shown is theviability of the multiple myeloma cell line, ARH-77, after the silencingof Gadd45β or MKK7 using three different Gadd45β-specific sh-RNAs (i.e.sh-Gadd45β-1, sh-Gadd45β-2, or sh-Gadd45β-3), two differentMKK7-specific sh-RNAs (i.e. sh-MKK7-1 or sh-MKK7-2), and two differentnon-specific sh-RNAs (i.e. sh-NS-1 or sh-NS-2). (A, B) Multiple myelomacell lines were infected with the indicated sh-RNA-expressingpLentiLox.3.7 lentivirus, then eGFP⁺ multiple myeloma cells (that iscells infected with lentivirus) were sorted using a BD FACSAria™ II cellsorter as in FIG. 16. [³H]Thymidine incorporation assays were performed10 days after cell sorting, corresponding to day 8 in FIG. 16. Shown isthe percent of [³H]thymidine incorporation (that is c.p.m.), a measureof cell proliferation, at day 8 (that is 10 days after cell sorting)relative to the [³H]thymidine incorporation occurring in the same cellsat day 0 (that is 2 days after cell sorting). These data furtherestablish the target specificity of Z-DTPs for the Gadd45β/MKK7 complexin multiple myeloma cells (see also FIGS. 7, 8, 9 and 12, Z-DTP-inducedkilling and Gadd45β expression; FIG. 16, Gadd45β and MKK7 genesilencing), and confirm the essential role that Gadd45β plays inmultiple myeloma cell survival. Together, they also furthervalidate=Gadd45β as therapeutic target in multiple myeloma.

FIG. 18. (A, B, C) PI nuclear staining assays showing that thesh-RNA-mediated silencing of Gadd45β induces apoptosis in theZ-DTP-sensitive multiple myeloma cell lines, ARH-77 (FIG. 18A) andNCI-H929 (FIG. 18B), but not in the Z-DTP-resistant multiple myelomacell line, RPMI-8226 (FIG. 18C) (see also FIGS. 16 and 17,sh-RNA-mediated silencing; FIGS. 7, 8, and 12, multiple myeloma cellline sensitivity to Z-DTP-induced killing and Gadd45β expression) (FIG.18A, FIG. 18B, FIG. 18C). No significant induction of apoptosis wasobserved in the same multiple myeloma cell lines in the absence oflentiviral infection (uninfected) or after expression of MKK7-specificsh-RNAs (i.e. sh-MKK7-1 and sh-MKK7-2) or non-specific sh-RNAs (i.e.sh-NS-1 and sh-NS-2). Multiple myeloma cell lines were infected withsh-RNA-expressing pLentiLox.3.7 lentiviruses, and eGFP⁺ multiple myelomacells (that is cells infected with lentivirus) were sorted using a BDFACSAria™ II cell sorter as in FIG. 16. PI nuclear staining assays wereperformed 10 days after cell sorting, corresponding to day 8 in FIG. 16.The percentages of apoptotic cells (that is cells exhibiting sub-G₁ DNAcontent) are depicted in the histograms. (FIG. 18A) Importantly, thelevels of apoptosis induced by the different Gadd45β-specific sh-RNAs(that is sh-Gadd45β-1, sh-Gadd45β-2, and sh-Gadd45β-3) correlate withthe levels of Gadd45β downregulation induced by each of theseGadd45β-specific sh-RNAs (data not shown). (FIG. 18A, FIG. 18B, FIG.18C) These data further establish the target specificity of Z-DTPs forthe Gadd45β/MKK7 complex in multiple myeloma cells (see also FIGS. 7, 8and 9, killing assays with Z-DTPs; FIG. 12, statistically significantcorrelation between Gadd45β expression and cancer cell sensitivity toZ-DTP-induced killing; FIGS. 16 and 17, induction of multiple myelomacell killing by the downregulation of Gadd45β, but not of MKK7), andconfirm the essential role that Gadd45β plays in multiple myeloma cellsurvival. Together, they further validate Gadd45β as a therapeutictarget in multiple myeloma.

FIG. 19. (A, B, C) PI nuclear staining assays showing that thesh-RNA-mediated silencing of either MKK7 or Gadd45β does not affectcell-cycle distribution in multiple myeloma cell lines. Therepresentative lentivirus-infected multiple myeloma cell linesshown—that is ARH-77 (FIG. 19A), NCI-H929 (FIG. 19B), and RPMI-8226(FIG. 19C)—are from the same experiment exhibited in FIG. 18.Differently from the data shown in FIGS. 18A, B and C (in which PIstaining profiles are represented in a logarithmic scale, whichhighlights apoptosis), PI staining (that is FL2-A) in this figure isrepresented in a linear scale, which highlights cell-cycle distribution.The percentages of multiple myeloma cells in the different phases of thecell cycle (that is G₁, S, and G₂/M) are depicted in the histograms.(FIG. 19 A, FIG. 19 B) Cell-cycle analyses could not be performed withGadd45β-specific sh-RNAs in the ARH-77 (FIG. 19 A) and NCI-H929 (FIG. 19B) multiple myeloma cell lines, due to the induction of massiveapoptosis in these cells (see FIGS. 18A and 18B).

FIG. 20. (A, B, C) The sh-RNA-mediated silencing of MKK7 renders therepresentative Z-DTP-sensitive cell line, ARH-77, resistant toZ-/mDTP-induced killing. [³H]Thymidine incorporation assays showing theIC₅₀s of D-isomer negative control tetrapeptide (Z-DNC) (A, B, C),Z-DTP1 (A), Z-DTP2 (B), or mDTP3 (C) in ARH-77 multiple myeloma cellsexpressing either MKK7-specific (sh-MKK7) or non-specific sh-RNAs(sh-NS). Treatments of ARH-77 cells with Z-DNC, Z-DTP1, Z-DTP2, or mDTP3were for 3 days. It can be seen that sh-NS-expressing ARH-77 cells arehighly sensitive to Z-/mDTP-induced killing—shown by the IC₅₀ values of1.4 μM (Z-DTP1; A), 302 nM (Z-DTP2; B), and 303 nM (mDTP3; C)—similar towhat is seen in the uninfected, parental ARH-77 cells (see Table IV).(A, B, C) In contrast, sh-MKK7-expressing ARH-77 cells have becomecompletely resistant to Z-/mDTP-induced killing—shown by the IC₅₀values>10 μM—similar to what is seen in Z-DNC-treated ARH-77 cells.IC₅₀s were calculated as described in the Examples, using increasingconcentrations of Z-DNC (A, B, C), Z-DTP1 (A), Z-DTP2 (B), and mDTP3(C), ranging from 0.01 to 10 μM. Reported in the graphs are thepercentages of the counts per minute (c.p.m.), a measure of cellproliferation, obtained with peptide treated cells relative to thec.p.m. values obtained with untreated cells. Similar data were obtainedwith additional Z-/mDTP-sensitive multiple myeloma cell lines, includingthe U266, KMS-11, and KMS-12 cell lines (data not shown). (A, B, C)These data demonstrate the very high target specificity of Z-/mDTPs forthe Gadd45β/MKK7 complex in multiple myeloma cells (see also FIG. 12,correlation between Gadd45β expression and cancer cell sensitivity toZ-DTP-induced killing).

FIG. 21. (A, B, C, D) The compounds of the invention do not bind toeither Gadd45β or MKK7 in isolation; rather they require for binding theformation of a Gadd45β/MKK7 complex, as determined in biacore assays.(A) Shown is the binding of Gadd45β to the kinase domain of MKK7(MKK7_(KD)) immobilized on a chip. Different concentrations of Gadd45β(ranging from 20 to 200 nM) were injected onto the chip where MKK7_(KD)had been previously immobilized. The dose-dependent binding of Gadd45βto MKK7_(KD) and the dissociation curves of the Gadd45β/MKK7_(KD)complex were recorded and an equilibrium dissociation constant (K_(D))value of 4.0±0.7 nM was determined by averaging the values determined bythe kinetic parameters of each individual curve. Briefly, thetermodinamic parameter of equilibrium dissociation constant (K_(D)) wascalculated considering the association (k_(a)) and dissociation phases(k_(d)) corresponding to an increase or decrease in the SPR signal(expressed as response units, RU) respectively. (B) Binding of MKK7_(KD)to Gadd45β, when Gadd45β was immobilized on the chip. Here the K_(D)values were determined by injecting MKK7_(KD) at differentconcentrations (ranging from 1 to 25 nM) onto a chip with immobilizedGadd45β. As in (A), the dose-response curves were recorded at all thetested concentrations of MKK7_(KD). From these analyses a K_(D) value of3.4±0.6 nM was obtained—which is very similar to the K_(D) valueobtained in (A). (C) The injection of mDTP3 onto a chip containingeither Gadd45β or MKK7_(KD) is shown. To determine whether mDTP3 bindsto Gadd45β and/or to MKK7_(KD), a solution containing mDTP3 at aconcentration ranging from 1 nM and 10 μM was injected onto a chipderivatized with either one or the other protein. As it can be see, nobinding of mDTP3 to either Gadd45β or MKK7 was recorded even at thehighest concentration of mDTP3 used (i.e. 10 μM). (D) Shown is thebinding of mDTP3 to a preformed Gadd45β/MKK7 complex. A 100 nMconcentration of Gadd45β was injected onto the chip derivatized withMKK7_(KD) (60 μL; contact time of 3 min). Gadd45β proteins were allowedto dissociate for about 10 min and when approximately 50% of Gadd45β wasstill bound to MKK7_(KD), mDTP3 was injected at the concentration ofeither 10 nM, 100 nM, or 1 μM. As it can be seen, when it was used at aconcentration equivalent to or lower than 100 nM, mDTP3 induced a rapiddissociation of the Gadd45β/MKK7_(KD) complex. As it can also be seen,Gadd45β/MKK7_(KD) complex formation was rapidly recovered after mDTP3was washed away. At higher concentrations (e.g. 1 μM), however, mDTP3afforded dose-response binding and dissociation curves, indicating thatit was binding to either Gadd45β and/or to MKK7_(KD) or to a complex ofthe two proteins. These data support the view that the DTPs do not bindto Gadd45β or MKK7 proteins in isolation; rather they bind to one and/orthe other protein or to a complex of the two proteins only when the twoproteins come in contact with each other.

NOTE ON NOMENCLATURE USED HEREIN

In various parts of this specification, compounds are refereed to by asignifying code such as LTP, DTP, LNC, DTP1 etc. Codes containing “NC”describe compounds which are negative controls not encompassed withinthe scope of the invention. Codes containing “TP” (which is anabbreviation of for tetra or tri-peptide/peptoids, although it should benoted that some of the compounds are based on di-peptide/peptiod motifs)are within the scope of the invention. The “L” or “D” prefix denotesresidues in the L or D optical configuration. A numeric suffix denotes aspecific numbered compound detailed elsewhere. The prefix “Z” as in“Z-DTP” denotes a benzyloxycarbonyl N-terminal group. The “m” prefix asin “mDTP” denotes any modification of a DTP aimed at improving cellularuptake, cellular activity, and/or PK profile, such as the removal of theN and/or C terminus (e.g. as in mDTP1), the removal of the Z group andof the Arg or Glu residues of Z-DTP2 as in mDTP2 and mDTP3, respectively(further examples are provided in FIG. 13).

DETAILED DESCRIPTION OF THE INVENTION

The strategy underlining the present invention arises from anunderstanding that NF-κB-JNK crosstalk also controls survival versusprogrammed death of cells including cancer cells which would otherwisehave died. Significantly, Gadd45β is up-regulated in cancerous cells inresponse to NF-κB activation and is expressed constitutively at highlevels in multiple myeloma cells and other tumours, including diffuselarge B-cell lymphoma, Burkitt's lymphoma, promonocytic leukaemia andother leukemias, as well as in some solid tumours, includinghepatocellular carcinoma, bladder cancer, brain and central nervoussystem cancer, breast cancer, head and neck cancer, lung cancer, andprostate cancer. The present invention is based on the strategy ofpromoting programmed cell death by delivering Gadd45β/MKK7-targetingcompounds that prevent NF-κB-JNK crosstalk thereby enhancing JNKcytotoxic signalling in cells. Products and methods of the presentinvention may be especially relevant to treatment of disorderscharacterised by aberrant up-regulation of Gadd45β. They are alsorelevant to diseases and disorders where Gadd45β may not necessarily beaberrantly up-regulated, but where NF-κB is aberrantly up-regulated oractivated and where an inductor of programmed cell death viaGadd45β-MKK7 signalling may provide a treatment.

Examples of these diseases featuring aberrant up-regulation oractivation of NF-κB and where an inductor of programmed cell death viaGadd45β-MKK7 signalling may provide a treatment include: haematologicalmalignancies (such as multiple myeloma, mantle cell lymphoma, MALTlymphoma, diffuse large B-cell lymphoma, Hodgkin's lymphoma,myelodysplastic syndrome, adult T-cell leukaemia (HTLV-1), chroniclymphocytic leukaemia, chronic myeloid leukaemia, acute myelogenicleukaemia, and acute lymphocytic leukaemia), solid tumours (such asbreast cancer, cervical cancer, renal cancer, lung cancer, colon cancer,liver cancer, oesophageal cancer, gastric cancer, laryngeal cancer,thyroid cancer, parathyroid cancer, bladder cancer, ovarian cancer,prostate cancer, pancreatic cancer and many other cancers), othercancers (such as melanoma, cylindroma, squamous cell carcinoma [skin,and head and neck], oral carcinoma, endometrial carcinoma,retinoblastoma, astrocytoma, and glioblastoma), and other diseases anddisorders such as autoimmune diseases, chronic inflammatory diseases,degenerative diseases, ischemic diseases, and vascular diseases.

A broad range of diseases and disorders depend on the activity of NF-κB.Indeed, the pathogenesis of virtually every known human disease ordisorder is now being considered to depend on inflammation, and hence toinvolve NF-κB. This functions as a masterswitch of the inflammatoryresponse, coordinating expression of an array of over 200 genes encodingcytokines, receptors, transcription factors, chemokines,pro-inflammatory enzymes, and other factors, including pro-survivalfactors, which initiate and sustain inflammation. The compounds of theinvention inhibit the discrete pro-survival activity of NF-κB ininflammation. Therefore, diseases and disorders amenable to treatmentwith these compounds include, apart from conventional chronicinflammatory diseases (such as inflammatory bowel disease, rheumatoidarthritis, and psoriasis), other diseases and disorders that depend on asignificant inflammatory component. Examples of such diseases anddisorders, which are being treated with anti-inflammatory agents orNF-κB-inhibiting agents or have been proposed as suitable for treatmentwith NF-κB inhibitors and could also be treated with a compound of theinvention, include:

1. respiratory tract: obstructive diseases of the airways including:asthma, including bronchial, allergic, intrinsic, extrinsic,exercise-induced, drug-induced (including aspirin and NSAID-induced) anddust-induced asthma, both intermittent and persistent and of allseverities, and other causes of airway hyper-responsiveness; chronicobstructive pulmonary disease (COPD); bronchitis, including infectiousand eosinophilic bronchitis; emphysema; bronchiectasis; cystic fibrosis;sarcoidosis; farmer's lung and related diseases; hypersensitivitypneumonitis; lung fibrosis, including cryptogenic fibrosing alveolitis,idiopathic interstitial pneumonias, fibrosis complicatinganti-neoplastic therapy and chronic infection, including tuberculosisand aspergillosis and other fungal infections; complications of lungtransplantation; vasculitic and thrombotic disorders of the lungvasculature, and pulmonary hypertension; antitussive activity includingtreatment of chronic cough associated with inflammatory and secretoryconditions of the airways, and iatrogenic cough; acute and chronicrhinitis including rhinitis medicamentosa, and vasomotor rhinitis;perennial and seasonal allergic rhinitis including rhinitis nervosa (hayfever); nasal polyposis; acute viral infection including the commoncold, and infection due to respiratory syncytial virus, influenza,coronavirus (including SARS) or adenovirus; or eosinophilic esophagitis;2. bone and joints: arthritides associated with or includingosteoarthritis/osteoarthrosis, both primary and secondary to, forexample, congenital hip dysplasia; cervical and lumbar spondylitis, andlow back and neck pain; osteoporosis; rheumatoid arthritis and Still'sdisease; seronegative spondyloarthropathies including ankylosingspondylitis, psoriatic arthritis, reactive arthritis andundifferentiated spondarthropathy; septic arthritis and otherinfection-related arthopathies and bone disorders such as tuberculosis,including Potts' disease and Poncet's syndrome; acute and chroniccrystal-induced synovitis including urate gout, calcium pyrophosphatedeposition disease, and calcium apatite related tendon, bursal andsynovial inflammation; Behcet's disease; primary and secondary Sjogren'ssyndrome; systemic sclerosis and limited scleroderma; systemic lupuserythematosus, mixed connective tissue disease, and undifferentiatedconnective tissue disease; inflammatory myopathies includingdermatomyositits and polymyositis; polymalgia rheumatica; juvenilearthritis including idiopathic inflammatory arthritides of whateverjoint distribution and associated syndromes, and rheumatic fever and itssystemic complications; vasculitides including giant cell arteritis,Takayasu's arteritis, Churg-Strauss syndrome, polyarteritis nodosa,microscopic polyarteritis, and vasculitides associated with viralinfection, hypersensitivity reactions, cryoglobulins, and paraproteins;low back pain; Familial Mediterranean fever, Muckle-Wells syndrome, andFamilial Hibernian Fever, Kikuchi disease; drug-induced arthalgias,tendonititides, and myopathies;3. pain and connective tissue remodelling of musculoskeletal disordersdue to injury [for example sports injury] or disease: arthitides (forexample rheumatoid arthritis, osteoarthritis, gout or crystalarthropathy), other joint disease (such as intervertebral discdegeneration or temporomandibular joint degeneration), bone remodellingdisease (such as osteoporosis, Paget's disease or osteonecrosis),polychondritits, scleroderma, mixed connective tissue disorder,spondyloarthropathies or periodontal disease (such as periodontitis);4. skin: psoriasis, atopic dermatitis, contact dermatitis or othereczematous dermatoses, and delayed-type hypersensitivity reactions;phyto- and photodermatitis; seborrhoeic dermatitis, dermatitisherpetiformis, lichen planus, lichen sclerosus et atrophica, pyodermagangrenosum, skin sarcoid, discoid lupus erythematosus, pemphigus,pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitides,toxic erythemas, cutaneous eosinophilias, alopecia greata, male-patternbaldness, Sweet's syndrome, Weber-Christian syndrome, erythemamultiforme; cellulitis, both infective and non-infective; panniculitis;cutaneous lymphomas, non-melanoma skin cancer and other dysplasticlesions; drug-induced disorders including fixed drug eruptions;5. eyes: blepharitis; conjunctivitis, including perennial and vernalallergic conjunctivitis; iritis; anterior and posterior uveitis;choroiditis; autoimmune; degenerative or inflammatory disordersaffecting the retina; ophthalmitis including sympathetic ophthalmitis;sarcoidosis; infections including viral, fungal, and bacterial;6. gastrointestinal tract: glossitis, gingivitis, periodontitis;oesophagitis, including reflux; eosinophilic gastro-enteritis,mastocytosis, Crohn's disease, colitis including ulcerative colitis,proctitis, pruritis ani; coeliac disease, irritable bowel syndrome, andfood-related allergies which may have effects remote from the gut (forexample migraine, rhinitis or eczema);7. abdominal: hepatitis, including autoimmune, alcoholic and viral;fibrosis and cirrhosis of the liver; cholecystitis; pancreatitis, bothacute and chronic;8. genitourinary: nephritis including interstitial andglomerulonephritis; nephrotic syndrome; cystitis including acute andchronic (interstitial) cystitis and Hunner's ulcer; acute and chronicurethritis, prostatitis, epididymitis, oophoritis and salpingitis;vulvo-vaginitis; Peyronie's disease; erectile dysfunction (both male andfemale);9. allograft rejection: acute and chronic following, for example,transplantation of kidney, heart, liver, lung, bone marrow, skin orcornea or following blood transfusion; or chronic graft versus hostdisease;10. CNS: Atzheimer's disease and other dementing disorders including CJDand nvCJD; amyloidosis; multiple sclerosis and other demyelinatingsyndromes; cerebral atherosclerosis and vasculitis; temporal arteritis;myasthenia gravis; acute and chronic pain (acute, intermittent orpersistent, whether of central or peripheral origin) including visceralpain, headache, migraine, trigeminal neuralgia, atypical facial pain,joint and bone pain, pain arising from cancer and tumor invasion,neuropathic pain syndromes including diabetic, post-herpetic, andHIV-associated neuropathies; neurosarcoidosis; central and peripheralnervous system complications of malignant, infectious or autoimmuneprocesses;11. other auto-immune and allergic disorders including Hashimoto'sthyroiditis, Graves' disease, Addison's disease, diabetes mellitus,idiopathic thrombocytopaenic purpura, eosinophilic fasciitis, hyper-IgEsyndrome, antiphospholipid syndrome;12. other disorders with an inflammatory or immunological component;including acquired immune deficiency syndrome (AIDS), leprosy, Sezarysyndrome, and paraneoplastic syndromes;13. cardiovascular: atherosclerosis, affecting the coronary andperipheral circulation; pericarditis; myocarditis, inflammatory andauto-immune cardiomyopathies including myocardial sarcoid; ischaemicreperfusion injuries; endocarditis, valvulitis, and aortitis includinginfective (for example syphilitic); vasculitides; disorders of theproximal and peripheral veins including phlebitis and thrombosis,including deep vein thrombosis and complications of varicose veins;14. gastrointestinal tract: Coeliac disease, proctitis, eosinopilicgastro-enteritis, mastocytosis, Crohn's disease, ulcerative colitis,microscopic colitis, indeterminant colitis, irritable bowel disorder,irritable bowel syndrome, non-inflammatory diarrhea, food-relatedallergies which have effects remote from the gut, e.g., migraine,rhinitis and eczema.

To this end the inventors have developed a number of synthetic moleculesbased on D-enantiomers of tetrapeptides, tripeptides, dipeptides andsimilar peptide-mimetics including peptoid moeties that disrupt theGadd45β/MKK7 interaction. Importantly, these compounds show Gadd45βinhibitory activity without suppressing MKK7 kinase function. This isimportant because it confirms that the compounds of the invention caninduce JNK cytotoxic signalling via inhibition of Gadd45β/MKK7complexes.

The synthetic molecules do not bind Gadd45β nor MKK7 in isolation, butthey bind to one or another protein when the proteins are in contactwith each other in the bound or unbound state, presumably by recognizinga surface that becomes available on Gadd45β, MKK7, and/or a complex ofthe two proteins only when Gadd45β and MKK7 come in contact with eachother, and consequently inducing a conformational modification in one ofthe two proteins or in the complex as whole that triggers thedissociation of the complex. This property is of particular interest,since it ensures that the compounds have a very high specificity for thetarget (i.e. the Gadd45β/MKK7 complex) and reduce the probability thatthe compounds of the invention can interact and so affect proteins thathave a structure similar to that of Gadd45β or MKK7. This property—whichestablish that the therapeutic target of the compounds of the inventionis the interface between two proteins (i.e. Gadd45β and MKK7)—alsoensures that the compounds of the invention will not block the globalbiological activities of Gadd45β or MKK7 in vivo, but rather willselectively interfere with the biological functions that Gadd45β or MKK7have as part of the Gadd45β/MKK7 complex.

Remarkably, compounds of the invention have been shown to induceapoptosis in multiple myeloma cell lines and primary tumour cells, andother tumour B-cell lines, including diffuse large B-cell lymphoma andBurkitt's lymphoma cell lines, as well as other cancers such aspromonocytic leukaemia, with IC₅₀s in the low nanomolar range, but tohave no activity on tumour T-cell lines or on normal cells such asuntransformed fibroblasts, bone marrow stromal cells (BMSCs), peripheralblood mononuclear cells (PBMNCs), and mesenkymal stem cells (MSCs), orin purified primary B- and T-lymphocytes from mice, even when used atvery high concentrations (that is 100 μM). This is evidence for theirhaving specificity in their cytotoxic activity for cells with abnormallyconstitutively active NF-κB. Importantly, compounds of the invention areresistant to proteolysis, soluble and stable in biological fluidsretaining full inhibitory activity after prolonged incubation with humanserum and therefore appear suitable candidates for systemic use.

The compounds of the invention show high target specificity for theGadd45β/MKK7 complex in cells. This is shown by the findings that: 1) Ina large panel of tumour cell lines there is a highly significantstatistical correlation between levels of Gadd45β expression and cancercell sensitivity to Z-/mDTP-induced killing; 2) sh-RNA-mediateddownregulation of Gadd45β induces apoptosis in Z-/mDTP-sensitive but notin Z-/mDTP-resistant cancer cell lines, and the kinetics of apoptosisinduction by Gadd45β-specific sh-RNAs in these cell lines is similar tothose observed with Z-/mDTPs; 3) the sh-RNA-mediated downregulation ofMKK7 renders Z-/mDTP-sensitive cancer cell lines completely resistant toZ-/mDTP-induced killing; 4) the therapeutic target of the invention isthe interface between two proteins, Gadd45β and MKK7—which furtherprovides potential for high target selectivity, a key advantage of oursolution over existing therapies. These data, together with the lowtoxicity of Z-/mDTPs to normal cells and the findings that knockoutablation of Gadd45β is well tolerated in mice, indicate that targetingthe discreet pro-survival functions of NF-κB in cell survival viaZ-/mDTP-mediated inhibition of Gadd45β/MKK7 can provide a therapy thatis more specific, less toxic, and hence more effective than therapiestargeting the NF-κB pathway and/or the proteasome.

Furthermore, compounds of the invention have no toxicity to normal cellsand inhibition of Gadd45β appears to have no or few side effects becauseGadd45β knock-out mice are viable and apparently healthy, indicatingthat complete Gadd45β inactivation is well tolerated in vivo. Compoundsof the invention are also stable, soluble, cell-permeable and thereforesuitable for the treatment of multiple myeloma, diffuse large B-celllymphoma and other cancers that depend on NF-κB for their survival. Theyare also useful for the treatment of chronic inflammatory and autoimmunediseases especially those mediated by NF-κB. Compounds of the inventionalso have PK profiles which are attractive for therapeutic use.

The invention also relates to the development of clinically usefulassays to predict Z-/mDTP therapy response in patients. The data with alarge panel of tumour cell lines show that sensitivity toZ-/mDTP-induced killing correlates with a high degree of significancewith Gadd45β expression levels (p<0.01), thus establishing the highspecificity of Z-/mDTPs' cytotoxic action for Gadd45β. Furthermore,knocking down Gadd45β induces apoptosis in multiple myeloma cells,whereas knocking down MKK7 renders these cells completely resistant toZ-/mDTP-induced killing Together, these data indicate that, shouldZ-/mDTP therapy enter the clinic, it will be possible to predict patientresponder populations via simple and cost-effective qRT-PCR analysis.

According to a first aspect of the invention there is provided acompound of formula I:X₁-A-X₂  I:

-   -   wherein,    -   A is A″″,        -   or A″-[M-A′-]_(n) M-A′″;    -   A″ is A″,        -   A′″        -   or Z₁—Y₂—Y₃—Z₄, wherein Y₂—Y₃ is an oligopeptide moiety or            an oligopeptoid moiety having the residues Y₂—Y₃ and Z₁ is            attached to the N-terminal nitrogen of Y₂—Y₃ and Z₄ is            attached to the C-terminal carbon of Y₂—Y₃;    -   A″ is A′,        -   or Y₁—Y₂—Y₃—Z₄, wherein Y₁—Y₂—Y₃ is an oligopeptoid moiety            or an oligopeptoid moiety comprising the residues: Y₁—Y₂—Y₃            and Z₄ is attached to the C-terminal carbon of Y₁—Y₂—Y₃;    -   A′″ is A′,        -   or Z₁—Y₂—Y₃—Y₄, wherein Y₂—Y₃—Y₄ is an oligopeptoid moiety            or an oligopeptoid moiety comprising the residues Y₂—Y₃—Y₄            and Z₁ is attached to the N-terminal nitrogen of Y₂—Y₃—Y₄;    -   each occurrence of A′ is independently an oligopeptide moiety or        an oligopeptoid moiety comprising the residues Y₁—Y₂—Y₃—Y₄;    -   n is an integer from 0 to 18    -   Y₁ and Y₄ are independently amino acid residues or residues of        amino acid derivatives having aromatic side chains; according to        certain embodiments each side chain comprises an alkylene group        of from one to three carbons which is substituted once or twice        with a 5 to 10 membered carbocyclic or heterocyclic aromatic        group and optionally further substituted by alkyl of from 1 to 4        carbon atoms; said aromatic group is optionally substituted by        at least one substituent selected from hydroxyl, halogen or C1        to C4 alkyl or C1 to C4 alkoxy.    -   Y₂ is absent or is an amino acid residue or a residue of an        amino acid derivative preferably any of the 20 natural amino        acids in the L or D configuration and/or preferably an amino        acid residue or a residue of an amino acid derivative having a        side chain carrying preferably a negative charge in aqueous        solution at pH7;    -   Y₃ is an amino acid residue or a residue of an amino acid        derivative preferably any of the 20 natural amino acids in the L        or D configuration and/or preferably an amino acid residue or a        residue of an amino acid derivative having a side chain carrying        preferably a positive charge in aqueous solution at pH7,    -   Where Y₂ and Y₃ are both present in certain embodiments they are        preferably such that a salt-bridge is able to form between the        respective positive and negative charges of the side chains        and/or are such that the distance between the aromatic centres        on Y₁ and Y₄, or on X₁ and X₄, or on X₁ and Y₄, or on Y₁ and X₄        is no higher than 10 or 20 Angstroms and no smaller than 3        Angstroms. Preferably the side chains of Y₂ and Y₃ consist of no        more than 30 atoms. Y₂ and Y₃ may be naturally occurring amino        acids or N-methyl-amino acids in the L- or D-configuration.        Z₁ is a group of formula II:

which is linked to the N-terminal nitrogen of Y₂,W is absent, or a oxygen, or a nitrogen, or an alkylene group of fromone to three carbons, which alkylene group of from one to three carbonsis optionally substituted by at least one substituent selected fromalkyl of from one to four carbons, or 5-10 membered carbocyclic orheterocyclic aromatic group;J is a 5-10 membered carbocyclic or heterocyclic aromatic group, whicharomatic group is optionally substituted by at least one substituentselected from hydroxyl, halogen, alkyl of from one to four carbons, oralkoxy of from one to four carbon atoms;Z₄ represents a group of formula III:

which is linked to the C-terminal carbon of Y₃,R is hydrogen or alkyl of from one to four carbons;W′ is absent or an alkylene group of from one to three carbons,which alkylene group of from one to three carbons is optionallysubstituted by at least one substituent selected from alkyl of from oneto four carbons, or 5-10 membered carbocyclic or heterocyclic aromaticgroup;J′ is a 3-10 membered aliphatic carbocyclic group or a 5-10 memberedcarbocyclic or heterocyclic aromatic group,which aliphatic or aromatic group is optionally substituted by at leastone substituent selected from hydroxyl, halogen, alkyl of from one tofour carbons, or alkoxy of from one to four carbon atoms;M is a peptide bond between preceding oligopeptide or oligopeptoidmoiety (A′, A″ or A′″) and following oligopeptide or oligopeptoid moiety(A′, A″ or A′″) or a linker moiety attached via an amide bond, an esterbond, an ether bond, or a thioether bond to the terminal carboxylicgroup of preceding oligopeptide or oligopeptoid moiety (A′, A″ or A′″)and via an amide bond, an ester bond, an ether bond, or a thioether bondto the terminal amino group of following oligopeptoid moiety (A′, A″ orA′″);X₁ is absent, or is a moiety added to the amino terminal of A in orderto block the free amino group;X₂ is absent or is a moiety added to the carboxyl terminal of A in orderto block the free carboxylic group;According to certain embodiments W is absent or an alkylene of from 1 to3 carbons.

Preferably X₁ and X₂ are moieties of no more than 30 (or more preferably20 or 10) atoms,

with the proviso that X₁ is absent if A comprises Z₁ and X₂ is absent ifA comprises Z₄ (i.e., if there are no free amino or carboxyl groups atthe termini of the molecule, X₁ and X₂ are not required);

or derivatives thereof, said derivatives being selected from the groupconsisting of:

-   -   a) oligomers or multimers of molecules of the compound of        formula I, said oligomers and multimers comprising two or more        molecules of the compound of formula I each linked to a common        scaffold moiety via an amide bond formed between an amino or        carboxylic acid group present in molecules of the compound of        formula I and an opposite amino or carboxylic acid group on a        scaffold moiety said scaffold moiety participating in at least 2        amide bonds,    -   b) derivatives comprising a molecule of the compound of formula        I or an oligomer or multimer thereof as defined above in part a)        conjugated via an amide bond, an ester bond, an ether bond or a        thioether bond to:        -   PEG,        -   PEG-based compounds,        -   cell-penetrating peptides,        -   fluorescent dyes,        -   biotin or other tag moiety,        -   fatty acids,        -   nanoparticles of discrete size,        -   or chelating ligands complexed with metallic or        -   radioactive ions.    -   c) derivatives comprising a molecule of the compound of formula        I or an oligomer or multimer thereof as defined in part a) which        has been modified by amidation, glycosylation, carbamylation,        acylation, sulfation, phosphorylation, cyclization, lipidation,        pegylation or linkage to a peptide or peptiod fusion partner to        make a fusion peptide or fusion peptiod.        and    -   d) salts and solvates of a molecule of the compound of formula I        or of a derivative thereof as defined in part a) or b) above.

According to certain embodiments:

-   -   Y₁ is D-tryptophan,        -   L-tryptophan,        -   D-tyrosine,        -   L-tyrosine,        -   D-3,3-diphenyl-alanine,        -   L-3,3-diphenyl-alanine,        -   D-H-3-(4-pyridyl) alanine,        -   L-H-3-(4-pyridyl) alanine,        -   D-H-3-(3-pyridyl) alanine,        -   L-H-3-(3-pyridyl) alanine,        -   D-H-3-(2-pyridyl) alanine,        -   L-H-3-(2-pyridyl) alanine,        -   D-2-amino-4-phenyl-butirric acid,        -   L-2-amino-4-phenyl-butirric acid,        -   D-H-4-hydroxy-phenyl-glycine,        -   L-H-4-hydroxy-phenyl-glycine,        -   D-3-(2-furyl)-alanine,        -   L-3-(2-furyl)-alanine,        -   L-homoPhenylalanine,        -   D-homoPhenylalanine,        -   D-3-(4-quinolyl)-alanine,        -   L-3-(4-quinolyl)-alanine;        -   D-naphtyl-alanine        -   L-naphtyl-alanine        -   p-hydroxy-Benzoic acid        -   p-hydroxy-phenyl-acetic-acid        -   3-(p-hydroxy-phenyl)-propionic-acid        -   or N-methyl-derivatives in L- or D-configuration of any            above

Alternatively Y₁ may be:

-   -   D-phenylalanine,    -   L-phenylalanine,    -   D-tryptophan,    -   L-tryptophan,    -   D-tyrosine,    -   L-tyrosine,    -   D-3,3-diphenyl-alanine,    -   L-3,3-diphenyl-alanine,    -   D-H-3-(4-pyridyl) alanine,    -   L-H-3-(4-pyridyl) alanine,    -   D-H-3-(3-pyridyl) alanine,    -   L-H-3-(3-pyridyl) alanine,    -   D-H-3-(2-pyridyl) alanine,    -   L-H-3-(2-pyridyl) alanine,    -   D-2-amino-4-phenyl-butirric acid,    -   L-2-amino-4-phenyl-butirric acid,    -   D-phenyl-glycine,    -   L-phenyl-glycine,    -   D-H-4-hydroxy-phenyl-glycine,    -   L-H-4-hydroxy-phenyl-glycine,    -   D-3-(2-furyl)-alanine,    -   L-3-(2-furyl)-alanine,    -   L-Cyclohexylalanine,    -   D-Cyclohexylalanine,    -   L-homoPhenylalanine,    -   D-homoPhenylalanine,    -   D-3-(4-quinolyl)-alanine,    -   L-3-(4-quinolyl)-alanine;    -   D-naphtyl-alanine    -   or L-naphtyl-alanine

According to certain embodiments:

-   -   Y₂ is absent        -   D-glutamic acid,        -   L-glutamic acid,        -   D-aspartic acid,        -   L-aspartic acid,        -   L-Leucine        -   D-Leucine        -   L-Glutamine        -   D-Glutamine        -   L-Methionine        -   D-Methionine        -   D-2-amino-heptanedioic acid,        -   L-2-amino-heptanedioic acid,        -   a methyl or ethyl ester of any thereof,        -   L-homoserine,        -   D-homoserine;        -   or N-methyl-derivatives in L- or D-configuration of any            above

Alternatively Y₂ may be:

-   -   D-glutamic acid,    -   L-glutamic acid,    -   D-aspartic acid,    -   L-aspartic acid,    -   D-2-amino-heptanedioic acid,    -   L-2-amino-heptanedioic acid,    -   a methyl or ethyl ester of any thereof,    -   L-homoserine,    -   or D-homoserine;

According to certain embodiments:

-   -   Y₃ is D-arginine,        -   L-arginine,        -   L-Proline        -   D-Proline        -   D-histidine,        -   L-histidine,        -   D-lysine,        -   D-α,β-diaminopropionic acid (D-Dap),        -   L-α,β-diaminopropionic acid (L-Dap),        -   L-α,δ-diaminobutirric acid (L-Dab),        -   L-α,δ-diaminobutirric acid (L-Dab),        -   L-ornitine,        -   D-ornitine,        -   L-lysine;        -   or N-methyl-derivatives in L- or D-configuration of any            above

Alternatively Y₃ may be

-   -   D-arginine,    -   L-arginine,    -   D-histidine,    -   L-histidine,    -   D-lysine,    -   D-α,β-diaminopropionic acid (D-Dap),    -   L-α,β-diaminopropionic acid (L-Dap),    -   L-α,δ-diaminobutirric acid (L-Dab),    -   L-α,δ-diaminobutirric acid (L-Dab),    -   L-ornitine,    -   D-ornitine,    -   or L-lysine;

According to certain embodiments:

-   -   Y₄ is        -   D-phenylalanine,        -   L-phenylalanine,        -   D-tryptophan,        -   L-tryptophan,        -   D-tyrosine,        -   L-tyrosine,        -   D-3,3-diphenyl-alanine,        -   L-3,3-diphenyl-alanine,        -   D-H-3-(4-pyridyl) alanine,        -   L-H-3-(4-pyridyl) alanine,        -   D-H-3-(3-pyridyl) alanine,        -   L-H-3-(3-pyridyl) alanine,        -   D-H-3-(2-pyridyl) alanine,        -   L-H-3-(2-pyridyl) alanine,        -   D-2-amino-4-phenyl-butirric acid,        -   L-2-amino-4-phenyl-butirric acid,        -   D-phenyl-glycine,        -   L-phenyl-glycine,        -   D-H-4-hydroxy-phenyl-glycine,        -   L-H-4-hydroxy-phenyl-glycine,        -   D-3-(2-furyl)-alanine,        -   L-3-(2-furyl)-alanine,        -   L-homoPhenylalanine,        -   D-homoPhenylalanine,        -   D-3-(4-quinolyl)-alanine,        -   L-3-(4-quinolyl)-alanine;        -   D-naphtyl-alanine        -   L-naphtyl-alanine        -   Their N-methyl-derivatives in L- or        -   D-configuration        -   aniline        -   benzylamine        -   or 2-phenyl-ethyl-amine

Alternatively Y₄ may be

-   -   D-phenylalanine,    -   L-phenylalanine,    -   D-tryptophan,    -   L-tryptophan,    -   D-tyrosine,    -   L-tyrosine,    -   D-3,3-diphenyl-alanine,    -   L-3,3-diphenyl-alanine,    -   D-H-3-(4-pyridyl) alanine,    -   L-H-3-(4-pyridyl) alanine,    -   D-H-3-(3-pyridyl) alanine,    -   L-H-3-(3-pyridyl) alanine,    -   D-H-3-(2-pyridyl) alanine,    -   L-H-3-(2-pyridyl) alanine,    -   D-2-amino-4-phenyl-butirric acid,    -   L-2-amino-4-phenyl-butirric acid,    -   D-phenyl-glycine,    -   L-phenyl-glycine,    -   D-H-4-hydroxy-phenyl-glycine,    -   L-H-4-hydroxy-phenyl-glycine,    -   D-3-(2-furyl)-alanine,    -   L-3-(2-furyl)-alanine,    -   L-Cyclohexylalanine,    -   D-Cyclohexylalanine,    -   L-homoPhenylalanine,    -   D-homoPhenylalanine,    -   D-3-(4-quinolyl)-alanine,    -   L-3-(4-quinolyl)-alanine;    -   D-naphtyl-alanine    -   or L-naphtyl-alanine

According to certain preferred embodiments Y₁, Y₂, Y₃ and Y₄ are all asdescribed above. According to certain embodiments Y₁, Y₂, Y₃ and Y₄ areall described above with the proviso that Y₂ is

-   -   D-glutamic acid,    -   L-glutamic acid,    -   D-aspartic acid,    -   L-aspartic acid,    -   D-2-amino-heptanedioic acid,    -   L-2-amino-heptanedioic acid,    -   a methyl or ethyl ester of any thereof;    -   L-homoserine,    -   L-Leucine    -   D-Leucine    -   L-Glutamine    -   D-Glutamine    -   L-Methionine    -   D-Methionine    -   D-homoserine,    -   or N-methyl-derivatives in L- or D-configuration of any above        and Y₃ is    -   D-arginine,    -   L-arginine,    -   D-histidine,    -   L-histidine,    -   D-lysine,    -   L-lysine;    -   L-Proline    -   D-Proline    -   D-α,β-diaminopropionic acid (D-Dap),    -   L-α,β-diaminopropionic acid (L-Dap),    -   D-α,δ-diaminobutirric acid (D-Dab),    -   L-α,δ-diaminobutirric acid (L-Dab),    -   D-ornitine    -   L-ornitine    -   or N-methyl-derivatives in L- or D-configuration of any above

According to certain embodiments Y₁ and Y₂ are both as described abovebut one or both of Y₂ and Y₃ are absent. According to certainembodiments M is a peptide bond.

According to certain embodiments X₁ is a hydrogen or X₁ is one of thefollowing groups added to the amino terminal of the oligopeptidesequence so as to form an amide bond:

-   -   acetyl,    -   benzyloxycarbonyl,    -   2-chloro-benzyloxycarbonyl,    -   3-methoxy,4-hydroxy-benzoyl,    -   3-hydroxy,4-methoxy-benzoyl,    -   benzoyl,    -   or fluorenylmethoxycarbonyl;        X₂ is an hydroxyl group or is one of the following groups added        to the carbonyl acid terminal of the oligopeptide sequence so as        to form an amide bond:    -   amine,    -   D-phenylalanine,    -   L-phenylalanine,    -   D-tryptophan,    -   L-tryptophan,    -   D-tyrosine,    -   L-tyrosine    -   D-3,3-diphenyl-alanine,    -   L-3,3-diphenyl-alanine,    -   D-H-3-(4-pyridyl)-alanine,    -   L-H-3-(4-pyridyl)-alanine,    -   D-H-3-(3-pyridyl)-alanine,    -   L-H-3-(3-pyridyl)-alanine,    -   D-H-3-(2-pyridyl)-alanine,    -   L-H-3-(2-pyridyl)-alanine,    -   D-2-amino-4-phenyl-butirric acid,    -   L-2-amino-4-phenyl-butirric acid,    -   D-phenyl-glycine,    -   L-phenyl-glycine,    -   D-H-4-hydroxy-phenyl-glycine,    -   L-H-4-hydroxy-phenyl-glycine,    -   D-3-(2-furyl)-alanine,    -   L-3-(2-furyl)-alanine,    -   L-Cyclohexylalanine,    -   D-Cyclohexylalanine,    -   L-homoPhenylalanine,    -   D-homoPhenylalanine,    -   D-3-(4-quinolyl)-alanine,    -   L-3-(4-quinolyl)-alanine;    -   D-naphtyl-alanine    -   L-naphtyl-alanine    -   Or N-methyl-derivatives in L- or D-configuration of any above

According to certain embodiments:

-   -   Z₁ Is 4-hydroxy-benzoyl,        -   (4-hydroxy-phenyl)-acetyl        -   3-(4-hydroxy-phenyl)-propionyl        -   benzoyl,        -   benzyloxycarbonyl,        -   2-phenyl-acetyl        -   3-phenyl-propionyl        -   3,3-diphenyl-propionyl        -   3-(1H-Indol-3yl)-propionyl        -   (1H-Indol-3-yl)-acetyl        -   Furan-2-yl-acetyl        -   Furan-3-yl-acetyl        -   3-pyridin-4-yl-propionyl        -   3-pyridin-3-yl-propionyl        -   3-pyridin-2-yl-propionyl        -   3-pyrimidin-4-yl-propionyl        -   3-pyridazin-4-yl-propionyl        -   3-[1,3,5]Triazin-2-yl-propionyl        -   2-pyridin-4-yl-acetyl        -   2-pyridin-3-yl-acetyl        -   2-pyridin-2-yl-acetyl        -   2-pyrimidin-4-yl-acetyl        -   2-pyridazin-4-yl-acetyl        -   2-[1,3,5]Triazin-2-yl-acetyl        -   Naphthalen-1-yl-acetyl        -   Naphthalen-2-yl-acetyl        -   2-Naphthalen-1-yl-propionyl        -   or 2-Naphthalen-2-yl-propionyl    -   Y₂ is D-glutamic acid,        -   L-glutamic acid,        -   D-aspartic acid,        -   L-aspartic acid,        -   L-Leucine,        -   D-Leucine,        -   L-Glutamine,        -   D-Glutamine,        -   L-Methionine,        -   D-Methionine,        -   D-2-amino-heptanedioic acid,        -   L-2-amino-heptanedioic acid,        -   a methyl or ethyl ester of any thereof;        -   L-homoserine,        -   D-homoserine;        -   or N-methyl-derivatives in L- or D-configuration of any            above    -   Y₃ is D-arginine,        -   L-arginine,        -   D-histidine,        -   L-histidine,        -   L-proline,        -   D-proline,        -   D-lysine,        -   L-lysine;        -   D-α,β-diaminopropionic acid (D-Dap),        -   L-α,β-diaminopropionic acid (L-Dap),        -   D-α,δ-diaminobutirric acid (D-Dab),        -   L-α,δ-diaminobutirric acid (L-Dab),        -   D-ornitine        -   L-ornitine        -   or N-methyl-derivatives in L- or D-configuration of any            above    -   Z₄ is phenylamine,        -   benzylamine,        -   Phenetylamine        -   Cyclohexyl-amine        -   2-cyclohexyl-ethylamine        -   3-cyclohexyl-propylamine        -   4-(2-amino-ethyl)-phenol        -   4-amino-phenol        -   4-aminomethyl-phenol        -   1H-Indol-3-yl-amine        -   2-(1H-Indol-3-yl)-ethylamine        -   C-(1H-Indol-3-yl)-methylamine        -   2,2-diphenyl-ethylamine        -   2,2-dipyridin-4-yl-ethylamine        -   2-pyridin-4-yl-ethylamine        -   2-pyridin-3-yl-ethylamine        -   2-pyridin-2-yl-ethylamine        -   2-pyrimidin-4-yl-ethylamine        -   2-[1,3,5]Triazin-2-yl-ethylamine        -   C-furan-2-yl-methylamine        -   C-furan-3-yl-methylamine        -   or C-Naphthalen-2-yl-methylamine.

According to the convention all peptides and peptoids and regionsthereof are described from the N terminus to the C terminus.

n may be 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 or18. According to certain preferred embodiments n=0.

According to certain preferred embodiments A is A′. In such embodimentsthe compound is therefore essentially a tetrapeptide, a tripeptide, or adipeptide (or a corresponding peptoid) with optional blocking groups X₁and X₂ at one or more of the termini.

Oligopeptides

Oligopeptides are short polymers formed by the condensation of α-aminoacids (referred to herein as simply “amino acids”). The link between oneamino acid residue and the next is known as a peptide bond or an amidebond.

Amino-Acids

As used herein the term “amino acid” includes the 20 standard aminoacids (Isoleucine, Alanine, Leucine, Asparagine, Lysine, Aspartic Acid,Methionine, Cysteine, Phenylalanine, Glutamic Acid, Threonine,Glutamine, Tryptophan, Glycine, Valine, Proline, Serine, Tyrosine,Arginine and Histidine) in both their D and L optical configurations. Italso includes synthetic α-amino acids in both D and L forms. Accordingto certain embodiments the D configuration is preferred.

Amino Acid Derivatives

As used herein this term includes N-substituted glycines which differfrom α-amino acids in that their side chains are appended to nitrogenatoms along the molecule's backbone, rather than to the α-carbons (asthey are in amino acids). Also included in the term are methyl and ethylesters of α-amino acids, β-amino acids and N-methylated α-amino acids.

Oligopeptoids

Strictly speaking, the term “oligopeptide” relates to oligomers ofα-amino acids only. An analogous oligomer incorporating (at all or someresidue positions) an amino acid derivate (for example an N-substitutedglycine) is known as an oligopeptoid.

Derivatives

Preferably, derivatives of the compound of the first aspect of theinvention are functional derivatives. The term “functional derivative”is used herein to denote a chemical derivative of a compound of formula(I) having the same physiological function (as the correspondingunmodified compounds of formula (I) or alternatively having the same invitro function in a functional assay (for example, in one of the assaysdescribed in one of the examples disclosed herein).

Derivatives of the compound of the invention may comprise the structureof formula (I) modified by well known processes including amidation,glycosylation, carbamylation, acylation, for example acetylation,sulfation, phosphorylation, cyclization, lipidization and pegylation.The structure of formula (I) may be modified at random positions withinthe molecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties. Derivativesinclude compounds in which the N-terminal NH₂ group is replaced withanother group, for example a methoxy group. A compound of the inventionmay be a fusion protein, whereby the structure of formula (I) is fusedto another protein or polypeptide (the fusion partner) using methodsknown in the art. Any suitable peptide or protein can be used as thefusion partner (e.g., serum albumin, carbonic anhydrase,glutathione-S-transferase or thioredoxin, etc.). Preferred fusionpartners will not have an adverse biological activity in vivo. Suchfusion proteins may be made by linking the carboxy-terminus of thefusion partner to the amino-terminus of the structure of formula (I) orvice versa. Optionally, a cleavable linker may be used to link thestructure of formula (I) to the fusion partner. A resulting cleavablefusion protein may be cleaved in vivo such that an active form of acompound of the invention is released. Examples of such cleavablelinkers include, but are not limited to, the linkers D-D-D-D-Y [SEQ IDNO.: 227], G-P-R, A-G-G and H-P-F-H-L [SEQ ID NO.: 228], which can becleaved by enterokinase, thrombin, ubiquitin cleaving enzyme and renin,respectively. See, e.g., U.S. Pat. No. 6,410,707.

A compound of the invention may be a physiologically functionalderivative of the structure of formula (I). The term “physiologicallyfunctional derivative” is used herein to denote a chemical derivative ofa compound of formula (I) having the same physiological function as thecorresponding unmodified compound of formula (I). For example, aphysiologically functionally derivative may be convertible in the bodyto a compound of formula (I). According to the present invention,examples of physiologically functional derivatives include esters,amides, and carbamates; preferably esters and amides. Pharmaceuticallyacceptable esters and amides of the compounds of the invention maycomprise a C₁₋₂₀ alkyl-, C₂₋₂₀ alkenyl-, C₅₋₁₀ aryl-, C₅₋₁₀ or C₁₋₂₀alkyl-, or amino acid-ester or -amide attached at an appropriate site,for example at an acid group. Examples of suitable moieties arehydrophobic substituents with 4 to 26 carbon atoms, preferably 5 to 19carbon atoms. Suitable lipid groups include, but are not limited to, thefollowing: lauroyl (Ci₂H₂₃), palmityl (C₁₅H₃₁), oleyl (C₁₅H₂₉), stearyl(C₁₇H₃₅), cholate; and deoxycholate.

Methods for lipidization of sulfhydryl-containing compounds with fattyacid derivatives are disclosed in U.S. Pat. No. 5,936,092; U.S. Pat. No.6,093,692; and U.S. Pat. No. 6,225,445. Fatty acid derivatives of acompound of the invention comprising a compound of the invention linkedto fatty acid via a disulfide linkage may be used for delivery of acompound of the invention to neuronal cells and tissues. Lipidisationmarkedly increases the absorption of the compounds relative to the rateof absorption of the corresponding unlipidised compounds, as well asprolonging blood and tissue retention of the compounds. Moreover, thedisulfide linkage in lipidised derivative is relatively labile in thecells and thus facilitates intracellular release of the molecule fromthe fatty acid moieties. Suitable lipid-containing moieties arehydrophobic substituents with 4 to 26 carbon atoms, preferably 5 to 19carbon atoms. Suitable lipid groups include, but are not limited to, thefollowing: palmityl (C₁₅H₃₁), oleyl (C₁₅H₂₉), stearyl (C₁₇H₃₅), cholate;and deoxycholate.

Cyclization methods include cyclization through the formation of adisulfide bridge and head-to-tail cyclization using a cyclization resin.Cyclized peptides may have enhanced stability, including increasedresistance to enzymatic degradation, as a result of their conformationalconstraints. Cyclization may in particular be expedient where theuncyclized peptide includes an N-terminal cysteine group. Suitablecyclized peptides include monomeric and dimeric head-to-tail cyclizedstructures. Cyclized peptides may include one or more additionalresidues, especially an additional cysteine incorporated for the purposeof formation of a disulfide bond or a side chain incorporated for thepurpose of resin-based cyclization.

A compound of the invention may be a pegylated structure of formula (I).Pegylated compounds of the invention may provide additional advantagessuch as increased solubility, stability and circulating time of thepolypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337).

Chemical moieties for derivitization of a compound of the invention mayalso be selected from water soluble polymers such as polyethyleneglycol, ethylene glycol/propylene glycol copolymers,carboxymethylcellulose, dextran, polyvinyl alcohol and the like. Apolymer moiety for derivatisation of a compound of the invention may beof any molecular weight, and may be branched or unbranched. Polymers ofother molecular weights may be used, depending on the desiredtherapeutic profile, for example the duration of sustained releasedesired, the effects, if any on biological activity, the ease inhandling, the degree or lack of antigenicity and other known effects ofthe polyethylene glycol to a therapeutic protein or analog. For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

Salts and solvates of compounds of the invention that are suitable foruse in a medicament are those wherein a counterion or associated solventis pharmaceutically acceptable. However, salts and solvates havingnon-pharmaceutically acceptable counterions or associated solvents arewithin the scope of the present invention, for example, for use asintermediates in the preparation of the compounds of formula (I) andtheir pharmaceutically acceptable salts or solvates.

Suitable salts according to the invention include those formed withorganic or inorganic acids or bases. Pharmaceutically acceptable acidaddition salts include those formed with hydrochloric, hydrobromic,sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic,pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic,glycollic, lactic, salicylic, oxaloacetic, methanesulfonic,ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic,naphthalene-2-sulfonic, benzenesulfonic, and isetliionic acids. Otheracids such as oxalic, while not in themselves pharmaceuticallyacceptable, may be useful as intermediates in obtaining the compounds ofthe invention and their pharmaceutical acceptable salts.Pharmaceutically acceptable salts with bases include ammonium salts,alkali metal salts, for example potassium and sodium salts, alkalineearth metal salts, for example calcium and magnesium salts, and saltswith organic bases, for example dicyclohexylamine andN-methyl-D-glucomine.

Those skilled in the art of organic chemistry will appreciate that manyorganic compounds can form complexes with solvents in which they arereacted or from which they are precipitated or crystallized. Suchcomplexes are known as “solvates”. For example, a complex with water isknown as a “hydrate”. The present invention provides solvates ofcompounds of the invention.

According to certain preferred embodiments, the compound as a half-lifein the human circulation of at least 0.1, 0.2, 0.5, 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11 or most preferably at least 12 hours.

Preferably, the compound retains at least 20, 30, 40, 50, 60, 70, 80, 90or most preferably 99% of its capacity to bind to Gadd45β and/or MKK7(and/or an association of both) as assessed in an in vitro bindingassay, or at least 20, 30, 40, 50, 60, 70, 80, 90 or most preferably 99%of its capacity to block the Gadd45β interaction with MKK7 as assessedin an in vitro competitive binding assay following incubation in normalhuman serum for at 24 hours at 37 degrees Celsius.

Alternatively or additionally, the compound has at least one of thefollowing activities:

-   -   a) The ability to inhibit at least 20, 30, 40, 50, 60, 70, 80,        90 or most preferably 99% of the MKK7 interactions with Gadd45β        under the assay conditions described in the examples.    -   b) The ability in vitro to kill at least 20, 30, 40, 50, 60, 70,        80, 90 or most preferably 99% of cells in a culture of a human        myeloma cell line selected from the group consisting of U266,        KMS-11, NCI-H929, ARH-77, JJN-3, KMS-12, KMS-18, and KMS-27, or        of a culture of the DLBCL cell line LY-3, or of a culture of the        pro-monocytic cell line U937, or of a culture of the Burkitt's        lymphoma cell line BJAB or a culture of primary tumour cells        (for example primary multiple myeloma tumour cells) under        conditions in which at least 90% of the T-cell line JURKAT is        not killed.

According to certain preferred embodiments the oligopeptide core moietyof the compound, identified as A in Formula I has an amino acid sequenceselected from the group consisting of:

[SEQ ID NO.: 2] (L-Tyr)-(L-Asp)-(L-His)-(L-Phe), [SEQ ID NO.: 3](L-Tyr)-(L-Glu)-(L-Arg)-(L-Phe), [SEQ ID NO.: 4](L-Tyr)-(L-Glu)-(L-His)-(L-Phe), [SEQ ID NO.: 5](L-Trp)-(L-Asp)-(L-His)-(L-Phe), [SEQ ID NO.: 6](L-Trp)-(L-Glu)-(L-His)-(L-Phe), [SEQ ID NO.: 7](L-Tyr)-(L-Asp)-(L-Arg)-(L-Phe), [SEQ ID NO.: 8](L-Tyr)-(L-Asp)-(L-Lys)-(L-Phe), [SEQ ID NO.: 9](L-Tyr)-(L-Glu)-(L-Lys)-(L-Phe), [SEQ ID NO.: 10](L-Trp)-(L-Glu)-(L-Lys)-(L-Phe), [SEQ ID NO.: 11](L-Trp)-(L-Glu)-(L-Arg)-(L-Phe), [SEQ ID NO.: 12](L-Trp)-(L-Asp)-(L-Lys)-(L-Phe), [SEQ ID NO.: 13](L-Trp)-(L-Asp)-(L-Arg)-(L-Phe), [SEQ ID NO.: 14](L-Tyr)-(L-Asp)-(L-His)-(L-Trp), [SEQ ID NO.: 15](L-Tyr)-(L-Glu)-(L-His)-(L-Trp), [SEQ ID NO.: 16](L-Trp)-(L-Asp)-(L-His)-(L-Trp), [SEQ ID NO.: 17](L-Trp)-(L-Glu)-(L-His)-(L-Trp), [SEQ ID NO.: 18](L-Tyr)-(L-Asp)-(L-Arg)-(L-Trp), [SEQ ID NO.: 19](L-Tyr)-(L-Asp)-(L-Lys)-(L-Trp), [SEQ ID NO.: 20](L-Tyr)-(L-Glu)-(L-Lys)-(L-Trp), [SEQ ID NO.: 21](L-Tyr)-(L-Glu)-(L-Arg)-(L-Trp), [SEQ ID NO.: 22](L-Trp)-(L-Glu)-(L-Lys)-(L-Trp), [SEQ ID NO.: 23](L-Trp)-(L-Glu)-(L-Arg)-(L-Trp), [SEQ ID NO.: 24](L-Trp)-(L-Asp)-(L-Lys)-(L-Trp), [SEQ ID NO.: 25](L-Trp)-(L-Asp)-(L-Arg)-(L-Trp), [SEQ ID NO.: 26](L-Tyr)-(L-Asp)-(L-His)-(L-Tyr), [SEQ ID NO.: 27](D-Tyr)-(D-Glu)-(D-Arg)-(D-Phe), [SEQ ID NO.: 28](D-Tyr)-(D-Asp)-(D-His)-(D-Phe), [SEQ ID NO.: 29](D-Trp)-(D-Glu)-(D-Arg)-(D-Phe), [SEQ ID NO.: 30](D-Trp)-(D-Asp)-(D-His)-(D-Phe), [SEQ ID NO.: 31](D-Tyr)-(D-Asp)-(D-Arg)-(D-Phe), [SEQ ID NO.: 32](D-Tyr)-(D-Asp)-(D-His)-(D-Tyr), [SEQ ID NO.: 33](D-Tyr)-(D-Glu)-(D-Arg)-(D-Tyr), [SEQ ID NO.: 34](D-Trp)-(D-Asp)-(D-His)-(D-Typ), [SEQ ID NO.: 35](D-Trp)-(D-Glu)-(D-Arg)-(D-Typ), [SEQ ID NO.: 36](D-Tyr)-(D-Asp)-(D-Lys)-(D-Phe), [SEQ ID NO.: 208](D-Tyr)-(D-Glu)-(D-His)-(D-Phe), [SEQ ID NO.: 209](D-Tyr)-(D-Asp)-(D-Lys)-(D-Phe), [SEQ ID NO.: 210](D-Trp)-(D-Glu)-(D-His)-(D-Phe), [SEQ ID NO.: 211](D-Tyr)-(D-Glu)-(D-Lys)-(D-Phe), [SEQ ID NO.: 212](D-Trp)-(D-Glu)-(D-Lys)-(D-Phe), [SEQ ID NO.: 213](D-Trp)-(D-Asp)-(D-Lys)-(D-Phe), [SEQ ID NO.: 214](D-Tyr)-(D-Asp)-(D-His)-(D-Trp), [SEQ ID NO.: 215](D-Tyr)-(D-Glu)-(D-His)-(D-Trp), [SEQ ID NO.: 216](D-Trp)-(D-Asp)-(D-His)-(D-Trp), [SEQ ID NO.: 217](D-Trp)-(D-Glu)-(D-His)-(D-Trp), [SEQ ID NO.: 218](D-Tyr)-(D-Asp)-(D-Arg)-(D-Trp), [SEQ ID NO.: 219](D-Tyr)-(D-Asp)-(D-Lys)-(D-Trp), [SEQ ID NO.: 220](D-Tyr)-(D-Glu)-(D-Lys)-(D-Trp), [SEQ ID NO.: 221](D-Tyr)-(D-Glu)-(D-Arg)-(D-Trp), [SEQ ID NO.: 222](D-Trp)-(D-Glu)-(D-Lys)-(D-Trp), [SEQ ID NO.: 223](D-Trp)-(D-Glu)-(D-Arg)-(D-Trp), [SEQ ID NO.: 224](D-Trp)-(D-Asp)-(D-Lys)-(D-Trp), [SEQ ID NO.: 225](D-Trp)-(D-Gln)-(D-Arg)-(D-Trp), [SEQ ID NO.: 226](D-Trp)-(D-Asn)-(D-Lys)-(D-Trp), (L-Tyr)-(L-Asp)-(L-Phe),(D-Tyr)-(D-Asp)-(D-Phe), (L-Tyr)-(L-Glu)-(L-Phe),(L-Tyr)-(L-Arg)-(L-Phe), (D-Tyr)-(D-Arg)-(D-Phe),(D-Tyr)-(D-Glu)-(D-Phe), (D-Tyr)-(D-Pro)-(D-Phe)(D-Tyr)-(D-Leu)-(D-Phe), (D-Tyr)-(D-Asp)-(D-Tyr),(D-Tyr)-(D-Glu)-(D-Tyr), (D-Tyr)-(D-Arg)-(D-Tyr),(D-Tyr)-(D-Pro)-(D-Tyr), (D-Tyr)-(D-Leu)-(D-Tyr),(D-Phe)-(D-Pro)-(D-Phe) (D-Phe)-(D-Leu)-(D-Phe), (D-Phe)-(D-Arg)-(D-Tyr)(D-Phe)-(D-Glu)-(D-Tyr), (D-Phe)-(D-Asp)-(D-Tyr),(D-Phe)-(D-Pro)-(D-Tyr) (D-Phe)-(D-Leu)-(D-Tyr) (D-Tyr)-(D-Pro)-(D-Trp)(D-Tyr)-(D-Leu)-(D-Trp), (D-Tyr)-(D-Asp)-(D-Trp),(D-Tyr)-(D-Glu)-(D-Trp), (D-Tyr)-(D-Arg)-(D-Trp),(D-Tyr)-(D-Pro)-(D-Trp), (D-Tyr)-(D-Leu)-(D-Trp),(D-Phe)-(D-Pro)-(D-Trp) (D-Phe)-(D-Leu)-(D-Trp), (D-Phe)-(D-Arg)-(D-Trp)(D-Phe)-(D-Glu)-(D-Trp), (D-Phe)-(D-Asp)-(D-Trp),(D-Phe)-(D-Pro)-(D-Trp) and (D-Phe)-(D-Leu)-(D-Trp)

In other embodiments the A moiety is selected from the group consistingof:

-   p-hydroxybenzoic acid-(L-Glu)-(L-Arg)-aniline-   p-hydroxybenzoic acid-(D-Glu)-(L-Arg)-aniline-   p-hydroxybenzoic acid-(L-Glu)-(D-Arg)-aniline-   p-hydroxybenzoic acid-(D-Glu)-(D-Arg)-aniline-   p-hydroxybenzoic acid-(L-Glu)-(L-Arg)-benzylamine-   p-hydroxybenzoic acid-(D-Glu)-(L-Arg)-benzylamine-   p-hydroxybenzoic acid-(L-Glu)-(D-Arg)-benzylamine-   p-hydroxybenzoic acid-(D-Glu)-(D-Arg)-benzylamine-   p-hydroxybenzoic acid-(L-Glu)-(L-Arg)-2-phenyl-ethyl-amine-   p-hydroxybenzoic acid-(D-Glu)-(L-Arg)-2-phenyl-ethyl-amine-   p-hydroxybenzoic acid-(L-Glu)-(D-Arg)-2-phenyl-ethyl-amine-   p-hydroxybenzoic acid-(D-Glu)-(D-Arg)-2-phenyl-ethyl-amine-   2-(4-hydroxy-phenyl) acetic acid-(L-Glu)-(L-Arg)-aniline-   2-(4-hydroxy-phenyl) acetic acid-(D-Glu)-(L-Arg)-aniline-   2-(4-hydroxy-phenyl) acetic acid-(L-Glu)-(D-Arg)-aniline-   2-(4-hydroxy-phenyl) acetic acid-(D-Glu)-(D-Arg)-aniline-   2-(4-hydroxy-phenyl) acetic acid-(L-Glu)-(L-Arg)-benzylamine-   2-(4-hydroxy-phenyl) acetic acid-(D-Glu)-(L-Arg)-benzylamine-   2-(4-hydroxy-phenyl) acetic acid-(L-Glu)-(D-Arg)-benzylamine-   2-(4-hydroxy-phenyl acetic acid-(D-Glu)-(D-Arg)-benzylamine-   2-(4-hydroxy-phenyl) acetic    acid-(L-Glu)-(L-Arg)-2-phenyl-ethyl-amine-   2-(4-hydroxy-phenyl) acetic    acid-(D-Glu)-(L-Arg)-2-phenyl-ethyl-amine-   2-(4-hydroxy-phenyl) acetic    acid-(L-Glu)-(D-Arg)-2-phenyl-ethyl-amine-   2-(4-hydroxy-phenyl) acetic    acid-(D-Glu)-(D-Arg)-2-phenyl-ethyl-amine-   3-(4-hydroxy-phenyl) propionic acid-(L-Glu)-(L-Arg)-aniline-   3-(4-hydroxy-phenyl) propionic acid-(D-Glu)-(L-Arg)-aniline-   3-(4-hydroxy-phenyl) propionic acid-(L-Glu)-(D-Arg)-aniline-   3-(4-hydroxy-phenyl) propionic acid-(D-Glu)-(D-Arg)-aniline-   3-(4-hydroxy-phenyl) propionic acid-(L-Glu)-(L-Arg)-benzylamine-   3-(4-hydroxy-phenyl) propionic acid-(D-Glu)-(L-Arg)-benzylamine-   3-(4-hydroxy-phenyl) propionic acid-(L-Glu)-(D-Arg)-benzylamine-   3-(4-hydroxy-phenyl) propionic acid-(D-Glu)-(D-Arg)-benzylamine-   3-(4-hydroxy-phenyl) propionic    acid-(L-Glu)-(L-Arg)-2-phenyl-ethyl-amine-   3-(4-hydroxy-phenyl) propionic    acid-(D-Glu)-(L-Arg)-2-phenyl-ethyl-amine-   3-(4-hydroxy-phenyl) propionic    acid-(L-Glu)-(D-Arg)-2-phenyl-ethyl-amine-   3-(4-hydroxy-phenyl) propionic    acid-(D-Glu)-(D-Arg)-2-phenyl-ethyl-amine-   p-hydroxybenzoic acid-(L-Arg)-aniline-   p-hydroxybenzoic acid-(D-Arg)-aniline-   p-hydroxybenzoic acid-(L-Glu)-aniline-   p-hydroxybenzoic acid-(D-Glu)-aniline-   p-hydroxybenzoic acid-(L-Arg)-benzylamine-   p-hydroxybenzoic acid-(D-Arg)-benzylamine-   p-hydroxybenzoic acid-(L-Glu)-benzylamine-   p-hydroxybenzoic acid-(D-Glu)-benzylamine-   p-hydroxybenzoic acid-(L-Arg)-2-phenyl-ethyl-amine-   p-hydroxybenzoic acid-(D-Arg)-2-phenyl-ethyl-amine-   p-hydroxybenzoic acid-(D-Glu)-2-phenyl-ethyl-amine-   p-hydroxybenzoic acid-(L-Glu)-2-phenyl-ethyl-amine-   2-(4-hydroxy-phenyl) acetic acid-(L-Arg)-aniline-   2-(4-hydroxy-phenyl) acetic acid-(D-Arg)-aniline-   2-(4-hydroxy-phenyl) acetic acid-(L-Glu)-aniline-   2-(4-hydroxy-phenyl) acetic acid-(D-Glu)-aniline-   2-(4-hydroxy-phenyl) acetic acid-(D-Arg)-benzylamine-   2-(4-hydroxy-phenyl) acetic acid-(L-Arg)-benzylamine-   2-(4-hydroxy-phenyl) acetic acid-(D-Glu)-benzylamine-   2-(4-hydroxy-phenyl) acetic acid-(L-Glu)-benzylamine-   2-(4-hydroxy-phenyl) acetic acid-(L-Arg)-2-phenyl-ethyl-amine-   2-(4-hydroxy-phenyl) acetic acid-(D-Arg)-2-phenyl-ethyl-amine-   2-(4-hydroxy-phenyl) acetic acid-(L-Glu)-2-phenyl-ethyl-amine-   2-(4-hydroxy-phenyl) acetic acid-(D-Glu)-2-phenyl-ethyl-amine-   3-(4-hydroxy-phenyl) propionic acid-(L-Arg)-aniline-   3-(4-hydroxy-phenyl) propionic acid-(D-Arg)-aniline-   3-(4-hydroxy-phenyl) propionic acid-(L-Glu)-aniline-   3-(4-hydroxy-phenyl) propionic acid-(D-Glu)-aniline-   3-(4-hydroxy-phenyl) propionic acid-(L-Arg)-benzylamine-   3-(4-hydroxy-phenyl) propionic acid-(D-Arg)-benzylamine-   3-(4-hydroxy-phenyl) propionic acid-(L-Glu)-benzylamine-   3-(4-hydroxy-phenyl) propionic acid-(D-Glu)-benzylamine-   3-(4-hydroxy-phenyl) propionic acid-(L-Arg)-2-phenyl-ethyl-amine-   3-(4-hydroxy-phenyl) propionic acid-(D-Arg)-2-phenyl-ethyl-amine-   3-(4-hydroxy-phenyl) propionic acid-(L-Glu)-2-phenyl-ethyl-amine-   3-(4-hydroxy-phenyl) propionic acid-(D-Glu)-2-phenyl-ethyl-amine

Alternatively, the moiety labelled as A′ in Formula I may be anoligopeptide having an amino acid sequence selected from the grouplisted directly above.

According to certain embodiments the A′ moiety is a peptide or peptoidmoiety having the residuesXaa₁-Xaa₂-Xaa₃-Xaa₄ wherein:

-   -   Xaa₁ is L-Tyr, D-Tyr, N-methyl-L-Tyr, N-methyl-D-Tyr,        p-hydroxybenzoic acid, 2-(4-hydroxy-phenyl) acetic acid,        3-(4-hydroxy-phenyl) propionic acid or acetyl    -   Xaa₂ is L-Glu, D-Glu, L-Asp or D-Asp, N-methyl-L-Glu,        N-methyl-D-Glu, N-methyl-L-Asp, N-methyl-D-Asp, L-Pro, D-Pro,        N-methyl-L-Pro, N-methyl-D-Pro, L-Leu, D-Leu, N-methyl-L-Leu,        N-methyl-D-Leu, or absent    -   Xaa₃ is L-Arg, D-Arg, L-His or D-His, L-Lys, D-Lys,        N-methyl-L-Arg, N-methyl-D-Arg, N-methyl-L-His, N-methyl-D-His,        N-methyl-L-Lys, N-methyl-D-Lys, or absent; and    -   Xaa₄ is aniline, benzylamine, 2-phenyl-ethyl-amine, L-Phe or        D-Phe, N-methyl-L-Phe, N-methyl-D-Phe, L-Trp, D-Trp,        N-methyl-L-Trp, N-methyl-D-Trp.

According to certain embodiments either Xaa₂ or Xaa₃ are absent but notboth Xaa₂ and Xaa₃. According to other embodiments Xaa₂ and Xaa₃ areboth absent.

M may be simply an amide bond between adjacent peptide or peptoidmoieties. Alternatively, it may be a molecular moiety introduced as aspacer and attached to adjacent peptide or peptoid moieties by amidebonds.

M may be an additional amino acid. Preferably it is an additional aminoacid with a non-bulky side chain, for example glycine, alanine or serineor derivatives of any thereof. Alternatively M may be a non-amino acidmoiety, for example, c-aminocaproic acid, 3-amino-propionic acid,4-amino-butirric acid. Other moieties can be methyl-amine, ethyl-amine,propyl-amine, butyl-amine, methylene, di-methylene, tri-methylene ortetra-methylene. In all cases M should be such that its presence doesnot materially interfere with binding between the A′ moiety and Gadd45βand/or MKK7. The extent of potential interference may be assessed by useof an in vitro binding assay as disclosed herein.

Oligomers and Multimers

The first aspect of the invention encompasses, oligomers or multimers ofmolecules of the compound of formula I, said oligomers and multimerscomprising two or more molecules of the compound of formula I eachlinked to a common scaffold moiety via an amide bond formed between anamine or carboxylic acid group present in molecules of the compound offormula I and an opposite amino or carboxylic acid group on a scaffoldmoiety said scaffold moiety participating in at least 2 amide bonds.

According to certain embodiments the common scaffold may be the aminoacid lysine. Lysine is a tri-functional amino acid, having in additionto the functional groups which define it as an amino acid, an aminogroup on its side claim. This tri-functional nature allows it to formthree amide bonds with peptides, peptoids or similar molecules. Othertri-functional amino acids which may be used as a common scaffoldinclude D-α,β-diaminopropionic acid (D-Dap), L-α,β-diaminopropionic acid(L-Dap), L-α,δ-diaminobutirric acid (L-Dab), L-α,δ-diaminobutirric acid(L-Dab), and L-ornitine, D-ornitine. Other tri-functional non-standardamino acids may also be used in accordance with the invention. Thecommon scaffold may also comprise branched peptides, peptoids or similarmolecules which incorporate tri-functional amino acids within theirsequence and have at least three functionally active terminal groupsable to form amide bonds.

Cell-Penetrating Peptides.

According to certain embodiments the compounds of formula I areconjugated to a cell penetrating peptide (CPP).

Such peptides may be attached to a compound of formula I either via oneor more covalent bonds or by non-covalent associations.

CPPs may either directly penetrate the plasmalemma, for example the CPPmay be Tat or a derivative, a peptide derived from the Antennapediasequence, or a poly-arginine tag, a PTD-4 peptide, or a functionallyequivalent cell-permeable peptide (Ho A, Schwarze S R, Mermelstein S J,Waksman G, Dowdy S F 2001 Synthetic protein transduction domains:enhanced transduction potential in vitro and in vivo. Cancer Res61:474-477).

Alternatively, the CPP may enter the cell by mediating endocytosis orthrough mediating the formation of transitory membrane-spanningstructures. For a discussion of cell penetrating peptides, the reader isdirected to Wagstaff et al. (2006). Curr. Med. Chem. 13:171-1387 andreferences therein.

According to certain embodiments compounds of the invention may beconjugated to nano-particles (for example nano-Gold) in order to promotecellular uptake

Fluorescent Dyes, Tag Moieties and Lipidated Derivatives.

Compounds of formula I may be conjugated to fluorescent dyes in orderthat their penetration into target tissues or cells may be monitored.Fluorescent dyes may be obtained with amino groups (i.e., succinimides,isothiocyanates, hydrazines), carboxyl groups (i.e., carbodiimides),thiol groups (i.e., maleimides and acetyl bromides) and azide groupswhich may be used to selectively react with the peptide moieties ofcompounds of formula I. Examples of fluorescent dyes includefluoresceine and its derivates, rhodamine and its derivatives.

Compounds of formula I may be conjugated to nanoparticles of discretesize such those described in Chithrani D B, Mol Membr Biol. 2010 Oct. 7,(Epub ahead of print) with a discrete size of up to 100 nm, whereby thepeptides or their derivatives can be attached by a disulphide bridge toallow specific release within the reducing environment of the cytosol.Also peptide-nanoparticles conjugated via amide, ether, ester, thioetherbonds can be used for the same purpose given the low toxicity of thesecompounds. Nanoparticles will favour cell uptake as well as will providea mean to visualize and quantify cell uptake by fluorescence techniques(Schrand A M, Lin J B, Hens S C, Hussain S M., Nanoscale. 2010 Sep. 27,Epub ahead of print).

Tag moieties may be attached by similar means and similarly allow formonitoring of the success of targeting to tissues and cells.

Fatty acid derivatives of a compound of the invention comprising acompound of formula I linked to a fatty acid via a disulfide linkage maybe used for delivery of a compound of the invention to cells andtissues. Lipidisation markedly increases the absorption of the compoundsrelative to the rate of absorption of the corresponding unlipidisedcompounds, as well as prolonging blood and tissue retention of thecompounds. Moreover, the disulfide linkage in lipidised derivative isrelatively labile in the cells and thus facilitates intracellularrelease of the molecule from the fatty acid moieties. Suitablelipid-containing moieties are hydrophobic substituents with 4 to 26carbon atoms, preferably 5 to 19 carbon atoms. Suitable lipid groupsinclude, but are not limited to, the following: palmityl (C₁₅H₃₁), oleyl(C₁₅H₂₉), stearyl (C₁₇H₃₅), cholate; linolate, and deoxycholate.

Ion Conjugates

The invention also encompasses compounds of formula I functionallyattached to metallic or radioactive ions. This attachment is typicallyachieved by the conjugation of an ion chelating agent (for example EDTA)which is chelated with the ion. By such means radioactive ions (forexample ^(99m)Tc, ¹¹¹In, ⁶⁴Cu, ⁶⁷Cu, ⁸⁹Sr, ⁹⁰Y, ^(117m)Sn, ¹⁵³Sm, ¹⁸⁶Re,¹⁸⁸Re, or ¹⁷⁷Lu) may be delivered to target cells as radiotherapy.Non-radioactive metallic ions (for example ions of gadolinium) may beused as a NMR-detectable marker.

Salts and Solvates

Salts and solvates of compounds of the invention that are suitable foruse in a medicament are those wherein a counterion or associated solventis pharmaceutically acceptable. However, salts and solvates havingnon-pharmaceutically acceptable counterions or associated solvents arewithin the scope of the present invention, for example, for use asintermediates in the preparation of the compounds of formula (I) andtheir pharmaceutically acceptable salts or solvates.

Suitable salts according to the invention include those formed withorganic or inorganic acids or bases. Pharmaceutically acceptable acidaddition salts include those formed with hydrochloric, hydrobromic,sulphuric, nitric, citric, tartaric, acetic, phosphoric, lactic,pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic,glycollic, lactic, salicylic, oxaloacetic, methanesulfonic,ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic,naphthalene-2-sulfonic, benzenesulfonic, and isethionic acids. Otheracids such as oxalic, while not in themselves pharmaceuticallyacceptable, may be useful as intermediates in obtaining the compounds ofthe invention and their pharmaceutical acceptable salts.Pharmaceutically acceptable salts with bases include ammonium salts,alkali metal salts, for example potassium and sodium salts, alkalineearth metal salts, for example calcium and magnesium salts, and saltswith organic bases, for example dicyclohexylamine andN-methyl-D-glucosamine.

Those skilled in the art of organic chemistry will appreciate that manyorganic compounds can form complexes with solvents in which they arereacted or from which they are precipitated or crystallized. Suchcomplexes are known as “solvates”. For example, a complex with water isknown as a “hydrate”. The present invention provides solvates ofcompounds of the invention.

Examples of preferred molecules of formula I are given below. Where theL/D configuration of an amino acid residue is not specified, bothconfigurations are encompassed

-   Acetyl-Tyr-Glu-Arg-Phe-NH₂ [SEQ ID NO.: 37]-   para-hydroxybenzoic acid-Glu-Arg-aniline-   para-hydroxybenzoic acid-Glu-Arg-benzylamine-   para-hydroxybenzoic acid-Glu-Arg-2-phenyl-ethyl-amine-   2-(4-hydroxyphenyl) acetic acid-Glu-Arg-aniline-   2-(4-hydroxyphenyl) acetic acid-Glu-Arg-benzylamine-   2-(4-hydroxyphenyl) acetic acid-Glu-Arg-2-phenyl-ethyl-amine-   3-(4-hydroxyphenyl) acetic acid-Glu-Arg-3-aniline-   3-(4-hydroxyphenyl) acetic acid-Glu-Arg-benzylamine-   3-(4-hydroxyphenyl) acetic acid-Glu-Arg-2-phenyl-ethyl-amine-   Acetyl-Tyr-Asp-His-Phe-NH₂ [SEQ ID NO.: 38]-   para-hydroxybenzoic-acid-Asp-His-aniline-   para-hydroxybenzoic-acid-Asp-His-benzylamine-   para-hydroxybenzoic-acid-Asp-His-3-phenyl-propyl-amine-   2-(4-hydroxyphenyl) acetic acid-Asp-His-aniline-   2-(4-hydroxyphenyl) acetic acid-Asp-His-benzylamine-   2-(4-hydroxyphenyl) acetic acid-Asp-His-2-phenyl-ethyl-amine-   3-(4-hydroxyphenyl) propionic acid-Asp-His-aniline-   3-(4-hydroxyphenyl) propionic acid-Asp-His-benzylamine-   3-(4-hydroxyphenyl) propionic acid-Asp-His-2-phenyl-ethyl-amine-   Acetyl-Tyr-Asp-Lys-Phe-NH₂ [SEQ ID NO.: 39]-   Acetyl-Tyr-Glu-Lys-Phe-NH₂ [SEQ ID NO.: 40]-   Acetyl-Tyr-Glu-His-Phe-NH₂ [SEQ ID NO.: 41]-   Acetyl-Tyr-Asp-Arg-Phe-NH₂, [SEQ ID NO.: 42]-   Acetyl-Trp-Glu-His-Phe-NH₂, [SEQ ID NO.: 43]-   Acetyl-Trp-Glu-Lys-Phe-NH₂, [SEQ ID NO.: 44]-   Acetyl-Trp-Asp-His-Phe-NH₂, [SEQ ID NO.: 45]-   Acetyl-Trp-Asp-Lys-Phe-NH₂, [SEQ ID NO.: 46]-   Acetyl-Tyr-Glu-Arg-Tyr-NH₂ [SEQ ID NO.: 47]-   Acetyl-Tyr-Asp-Lys-Tyr-NH₂ [SEQ ID NO.: 48]-   Acetyl-Tyr-Glu-Lys-Tyr-NH₂ [SEQ ID NO.: 49]-   Acetyl-Tyr-Glu-His-Tyr-NH₂ [SEQ ID NO.: 50]-   Acetyl-Tyr-Asp-Arg-Tyr-NH₂, [SEQ ID NO.: 51]-   Acetyl-Trp-Glu-His-Tyr-NH₂, [SEQ ID NO.: 52]-   Acetyl-Trp-Glu-Lys-Tyr-NH₂, [SEQ ID NO.: 53]-   Acetyl-Trp-Asp-His-Tyr-NH₂, [SEQ ID NO.: 54]-   Acetyl-Trp-Asp-Lys-Tyr-NH₂, [SEQ ID NO.: 55]-   internal lactam of acetyl-Tyr-Glu-Lys-Phe-NH₂ [SEQ ID NO.: 56]-   Acetyl-Tyr-Gln-Arg-Phe-NH₂ [SEQ ID NO.: 57]-   Acetyl-Tyr-Met-Arg-Phe-NH₂ [SEQ ID NO.: 58]-   Acetyl-Tyr-Leu-Arg-Phe-NH₂ [SEQ ID NO.: 59]-   Acetyl-Tyr-Arg-Phe-NH₂,-   Acetyl-Tyr-Arg-Tyr-NH₂,-   Acetyl-Tyr-Glu-Phe-NH₂,-   Acetyl-Tyr-Glu-Tyr-NH₂,-   Acetyl-Tyr-Asp-Phe-NH₂,-   Acetyl-Tyr-Asp-Tyr-NH₂,-   Acetyl-Tyr-Pro-Phe-NH₂,-   Acetyl-Tyr-Lys-Phe-NH₂,-   Acetyl-Tyr-His-Phe-NH₂,-   H-Tyr-Arg-Phe-NH₂,-   H-Tyr-Arg-Tyr-NH₂,-   H-Tyr-Glu-Phe-NH₂,-   H-Tyr-Glu-Tyr-NH₂,-   H-Tyr-Asp-Phe-NH₂,-   H-Tyr-Asp-Tyr-NH₂,-   H-Tyr-Pro-Phe-NH₂,-   H-Tyr-Lys-Phe-NH₂,-   H-Tyr-His-Phe-NH₂,-   Benzyloxycarbonyl-Tyr-Arg-Phe-NH₂,-   Benzyloxycarbonyl-Tyr-Arg-Tyr-NH₂,-   Benzyloxycarbonyl-Tyr-Glu-Phe-NH₂,-   Benzyloxycarbonyl-Tyr-Glu-Tyr-NH₂,-   Benzyloxycarbonyl-Tyr-Asp-Phe-NH₂,-   Benzyloxycarbonyl-Tyr-Asp-Tyr-NH₂,-   Benzyloxycarbonyl-Tyr-Pro-Phe-NH₂,-   Benzyloxycarbonyl-Tyr-Lys-Phe-NH₂,-   Benzyloxycarbonyl-Tyr-His-Phe-NH₂,-   Benzyloxycarbonyl-Tyr-Glu-Arg-Phe-NH₂, [SEQ ID NO.: 60]-   Benzyloxycarbonyl-Tyr-Asp-His-Phe-NH₂, [SEQ ID NO.: 61]-   Benzyloxycarbonyl-Tyr-Asp(OMe)-His-Phe-NH₂, [SEQ ID NO.: 62]-   Benzyloxycarbonyl-Tyr-Arg-Phe-NH₂,-   Benzyloxycarbonyl-Tyr-Glu-Phe-NH₂,-   Benzyloxycarbonyl-(N-methyl)Tyr-(N-methyl)Arg-(N-methyl)Phe-NH₂,-   Benzyloxycarbonyl-(N-methyl)Tyr-Glu-(N-methyl)Phe-NH₂,-   Benzyloxycarbonyl-Tyr-(N-methyl)Arg-(N-methyl)Phe-NH₂,-   Benzyloxycarbonyl-(N-methyl)Tyr-(N-methyl)Arg-Phe-NH₂,-   Benzyloxycarbonyl-Tyr-Glu-(N-methyl)Phe-NH₂,-   Benzyloxycarbonyl-Tyr-(N-methyl)Glu-Phe-NH₂,-   Benzyloxycarbonyl-(N-methyl)Tyr-Glu-Phe-NH₂,-   Acetyl-(N-methyl)Tyr-(N-methyl)Arg-(N-methyl)Phe-NH₂,-   Acetyl-(N-methyl)Tyr-Glu-(N-methyl)Phe-NH₂,-   Acetyl-Tyr-(N-methyl)Arg-(N-methyl)Phe-NH₂,-   Acetyl-(N-methyl)Tyr-(N-methyl)Arg-Phe-NH₂,-   Acetyl-Tyr-Glu-(N-methyl)Phe-NH₂,-   Acetyl-Tyr-(N-methyl)Glu-Phe-NH₂,-   Acetyl-(N-methyl)Tyr-Glu-Phe-NH₂,-   H—(N-methyl)Tyr-(N-methyl)Arg-(N-methyl)Phe-NH₂,-   H—(N-methyl)Tyr-Glu-(N-methyl)Phe-NH₂,-   H-Tyr-(N-methyl)Arg-(N-methyl)Phe-NH₂,-   H—(N-methyl)Tyr-(N-methyl)Arg-Phe-NH₂,-   H-Tyr-Glu-(N-methyl)Phe-NH₂,-   H-Tyr-(N-methyl)Glu-Phe-NH₂,-   H—(N-methyl)Tyr-Glu-Phe-NH₂,-   Acetyl-Tyr-Glu-(β-homo)Phe-NH₂,-   Acetyl-Tyr-(β-homo)Glu-Phe-NH₂,-   Acetyl-(β-homo)Tyr-Glu-Phe-NH₂,-   Acetyl-Tyr-Phe-NH₂,-   Acetyl-Phe-Tyr-NH₂,-   Benzyloxycarbonyl-Tyr-Phe-NH₂,-   Benzyloxycarbonyl-Phe-Tyr-NH₂,-   H-Tyr-Phe-NH₂,-   H-Phe-Tyr-NH₂,-   (3-Methoxy,4-hydroxy-benzoyl)-Tyr-Glu-Arg-Phe-NH₂, [SEQ ID NO.: 63]-   (3-Methoxy,4-hydroxy-benzoyl)-Tyr-Asp-His-Phe-NH₂, [SEQ ID NO.: 64]-   (3-Methoxy,4-hydroxy-benzoyl)-Tyr-Asp(OMe)-His-Phe-NH₂, [SEQ ID NO.:    65]-   (3-Methoxy,4-hydroxy-benzoyl)-Tyr-Arg-Phe-NH₂,-   (3-Methoxy,4-hydroxy-benzoyl)-Tyr-Glu-Phe-NH₂,-   Fluorenylmethyloxycarbonyl-Tyr-Glu-Arg-Phe-NH₂, [SEQ ID NO.: 66]-   Fluorenylmethyloxycarbonyl-Tyr-Asp-His-Phe-NH₂, [SEQ ID NO.: 67]-   Fluorenylmethyloxycarbonyl-Tyr-Asp(OMe)-His-Phe-NH₂, [SEQ ID NO.:    68]-   Fluorenylmethyloxycarbonyl-Tyr-Asp(OMe)-His-Phe-NH₂ [SEQ ID NO.: 69]-   Fluorenylmethyloxycarbonyl-Tyr-Arg-Phe-NH₂,-   Fluorenylmethyloxycarbonyl-Tyr-Glu-Phe-NH₂,-   Myristyl-Tyr-Glu-Arg-Phe-NH₂, [SEQ ID NO.: 70]-   Myristyl-Tyr-Asp-His-Phe-NH₂, [SEQ ID NO.: 71]-   Myristyl-Tyr-Arg-Phe-NH₂,-   Myristyl-Tyr-Glu-Phe-NH₂,-   Myristyl-Tyr-Asp(OMe)-His-Phe-NH₂, [SEQ ID NO.: 72]-   Acetyl-Tyr-Glu-Arg-Phe-Gly-Tyr-Glu-Arg-Phe-NH₂, [SEQ ID NO.: 73]-   Acetyl-Tyr-Asp-His-Phe-Gly-Tyr-Asp-His-Phe-NH₂, [SEQ ID NO.: 74]-   Acetyl-Tyr-Arg-Phe-Gly-Tyr-Arg-Phe-NH₂, [SEQ ID NO.: 75]-   Acetyl-Tyr-Asp(OMe)-His-Phe-Gly-Tyr-Asp(OMe)-His-Phe-NH₂, [SEQ ID    NO.: 76]-   benzyloxycarbonyl-Tyr-Glu-Arg-Phe-Gly-Tyr-Glu-Arg-Phe-NH₂, [SEQ ID    NO.: 77]-   benzyloxycarbonyl-Tyr-Asp-His-Phe-Gly-Tyr-Asp-His-Phe-NH₂, [SEQ ID    NO.: 78]-   benzyloxycarbonyl-Tyr-Arg-Phe-Gly-Tyr-Arg-Phe-NH₂, [SEQ ID NO.: 79]-   benzyloxycarbonyl-Tyr-Asp(OMe)-His-Phe-Gly-Tyr-Asp(OMe)-His-Phe-NH₂,    [SEQ ID NO.: 80]

Further examples of compounds of the invention include:

According to certain embodiments compounds disclosed specificallyherein, including in the examples, are preferred compounds or arepreferred embodiments of the A′ moiety of formula I. The presentinvention contemplates the multimer versions or the specific compoundsexplicitly disclosed herein. For example the present inventioncontemplates the 3 or 4 residue peptide or peptoid moieties of thespecific compounds disclosed herein as corresponding to the A, A′, A″,A′″ or A″″ moiety of compounds of formula I.

Pharmaceutical Compositions

According to a second aspect of the invention, there is provided apharmaceutical composition comprising a compound according to the firstaspect of the invention and a pharmaceutically acceptable carrier.

While it is possible for the active ingredient to be administered alone,it is preferable for it to be present in a pharmaceutical formulation orcomposition. Accordingly, the invention provides a pharmaceuticalformulation comprising a compound of formula (I), or derivative thereof,or a salt or solvate thereof, as defined above and a pharmaceuticallyacceptable carrier. Pharmaceutical compositions of the invention maytake the form of a pharmaceutical formulation as described below.

The pharmaceutical formulations according to the invention include thosesuitable for oral, parenteral (including subcutaneous, intradermal,intramuscular, intravenous, and intraarticular), inhalation (includingfine particle dusts or mists which may be generated by means of varioustypes of metered does pressurized aerosols, nebulizers or insufflators),rectal and topical (including dermal, transdermal, transmucosal, buccal,sublingual, and intraocular) administration, although the most suitableroute may depend upon, for example, the condition and disorder of therecipient.

The formulations may conveniently be presented in unit dosage form andmay be prepared by any of the methods well known in the art of pharmacy.All methods include the step of bringing the active ingredient intoassociation with the carrier which constitutes one or more accessoryingredients. In general the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both and then, ifnecessary, shaping the product into the desired formulation.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous liquidor a non-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also bepresented as a bolus, electuary or paste. Various pharmaceuticallyacceptable carriers and their formulation are described in standardformulation treatises, e.g., Remington's Pharmaceutical Sciences by E.W. Martin. See also Wang, Y. J. and Hanson, M. A., Journal of ParenteralScience and Technology, Technical Report No. 10, Supp. 42:2S, 1988.

A tablet may be made by compression or moulding, optionally with one ormore accessory ingredients. Compressed tablets may be prepared bycompressing in a suitable machine the active ingredient in afree-flowing form such as a powder or granules, optionally mixed with abinder, lubricant, inert diluent, lubricating, surface active ordispersing agent. Moulded tablets may be made by moulding in a suitablemachine a mixture of the powdered compound moistened with an inertliquid diluent. The tablets may optionally be coated or scored and maybe formulated so as to provide slow or controlled release of the activeingredient therein. The present compounds can, for example, beadministered in a form suitable for immediate release or extendedrelease. Immediate release or extended release can be achieved by theuse of suitable pharmaceutical compositions comprising the presentcompounds, or, particularly in the case of extended release, by the useof devices such as subcutaneous implants or osmotic pumps. The presentcompounds can also be administered liposomally.

Preferably, compositions according to the invention are suitable forsubcutaneous administration, for example by injection.

Exemplary compositions for oral administration include suspensions whichcan contain, for example, microcrystalline cellulose for imparting bulk,alginic acid or sodium alginate as a suspending agent, methylcelluloseas a viscosity enhancer, and sweeteners or flavoring agents such asthose known in the art; and immediate release tablets which can contain,for example, microcrystalline cellulose, dicalcium phosphate, starch,magnesium stearate and/or lactose and/or other excipients, binders,extenders, disintegrants, diluents and lubricants such as those known inthe art. The compounds of formula (I) or variant, derivative, salt orsolvate thereof can also be delivered through the oral cavity bysublingual and/or buccal administration. Molded tablets, compressedtablets or freeze-dried tablets are exemplary forms which may be used.Exemplary compositions include those formulating the present compound(s)with fast dissolving diluents such as mannitol, lactose, sucrose and/orcyclodextrins. Also included in such formulations may be high molecularweight excipients such as celluloses (avicel) or polyethylene glycols(PEG). Such formulations can also include an excipient to aid mucosaladhesion such as hydroxy propyl cellulose (HPC), hydroxy propyl methylcellulose (HPMC), sodium carboxy methyl cellulose (SCMC), maleicanhydride copolymer (e.g., Gantrez), and agents to control release suchas polyacrylic copolymer (e.g. Carbopol 934). Lubricants, glidants,flavors, coloring agents and stabilizers may also be added for ease offabrication and use.

Formulations for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents. The formulations may be presented in unit-dose or multi-dosecontainers, for example sealed ampoules and vials, and may be stored ina freeze-dried (lyophilised) condition requiring only the addition ofthe sterile liquid carrier, for example saline or water-for-injection,immediately prior to use. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described. Exemplary compositions for parenteraladministration include injectable solutions or suspensions which cancontain, for example, suitable non-toxic, parenterally acceptablediluents or solvents, such as mannitol, 1,3-butanediol, water, Ringer'ssolution, an isotonic sodium chloride solution, or other suitabledispersing or wetting and suspending agents, including synthetic mono-or diglycerides, and fatty acids, including oleic acid, or Cremaphor. Anaqueous carrier may be, for example, an isotonic buffer solution at a pHof from about 3.0 to about 8.0, preferably at a pH of from about 3.5 toabout 7.4, for example from 3.5 to 6.0, for example from 3.5 to about5.0. Useful buffers include sodium citrate-citric acid and sodiumphosphate-phosphoric acid, and sodium acetate/acetic acid buffers. Thecomposition preferably does not include oxidizing agents and othercompounds that are known to be deleterious to the compound of formula Iand related molecules. Excipients that can be included are, forinstance, other proteins, such as human serum albumin or plasmapreparations. If desired, the pharmaceutical composition may alsocontain minor amounts of non-toxic auxiliary substances, such as wettingor emulsifying agents, preservatives, and pH buffering agents and thelike, for example sodium acetate or sorbitan monolaurate.

Exemplary compositions for nasal aerosol or inhalation administrationinclude solutions in saline, which can contain, for example, benzylalcohol or other suitable preservatives, absorption promoters to enhancebioavailability, and/or other solubilizing or dispersing agents such asthose known in the art. Conveniently in compositions for nasal aerosolor inhalation administration the compound of the invention is deliveredin the form of an aerosol spray presentation from a pressurized pack ora nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoro-methane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit can be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator can be formulatedto contain a powder mix of the compound and a suitable powder base, forexample lactose or starch. In one specific, non-limiting example, acompound of the invention is administered as an aerosol from a metereddose valve, through an aerosol adapter also known as an actuator.Optionally, a stabilizer is also included, and/or porous particles fordeep lung delivery are included (e.g., see U.S. Pat. No. 6,447,743).

Formulations for rectal administration may be presented as a retentionenema or a suppository with the usual carriers such as cocoa butter,synthetic glyceride esters or polyethylene glycol. Such carriers aretypically solid at ordinary temperatures, but liquefy and/or dissolve inthe rectal cavity to release the drug.

Formulations for topical administration in the mouth, for examplebuccally or sublingually, include lozenges comprising the activeingredient in a flavoured basis such as sucrose and acacia ortragacanth, and pastilles comprising the active ingredient in a basissuch as gelatin and glycerine or sucrose and acacia. Exemplarycompositions for topical administration include a topical carrier suchas Plastibase (mineral oil gelled with polyethylene).

Preferred unit dosage formulations are those containing an effectivedose, as hereinbefore recited, or an appropriate fraction thereof, ofthe active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above, the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents.

The compounds of the invention are also suitably administered assustained-release systems. Suitable examples of sustained-releasesystems of the invention include suitable polymeric materials, forexample semi-permeable polymer matrices in the form of shaped articles,e.g., films, or mirocapsules; suitable hydrophobic materials, forexample as an emulsion in an acceptable oil; or ion exchange resins; andsparingly soluble derivatives of the compound of the invention, forexample, a sparingly soluble salt. Sustained-release systems may beadministered orally; rectally; parenterally; intravaginally;intraperitoneally; topically, for example as a powder, ointment, gel,drop or transdermal patch; bucally; or as an oral or nasal spray.

Preparations for administration can be suitably formulated to givecontrolled release of compounds of the invention. For example, thepharmaceutical compositions may be in the form of particles comprisingone or more of biodegradable polymers, polysaccharide jellifying and/orbioadhesive polymers, amphiphilic polymers, agents capable of modifyingthe interface properties of the particles of the compound of formula(I). These compositions exhibit certain biocompatibility features whichallow a controlled release of the active substance. See U.S. Pat. No.5,700,486.

A compound of the invention may be delivered by way of a pump (seeLanger, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:201, 1987; Buchwaldet al., Surgery 88:507, 1980; Saudek et al., N. Engl. J. Med. 321:574,1989) or by a continuous subcutaneous infusions, for example, using amini-pump. An intravenous bag solution may also be employed. Othercontrolled release systems are discussed in the review by Langer(Science 249:1527-1533, 1990). In another aspect of the disclosure,compounds of the invention are delivered by way of an implanted pump,described, for example, in U.S. Pat. No. 6,436,091; U.S. Pat. No.5,939,380; U.S. Pat. No. 5,993,414.

Implantable drug infusion devices are used to provide patients with aconstant and long term dosage or infusion of a drug or any othertherapeutic agent. Essentially such device may be categorized as eitheractive or passive. A compound of the present invention may be formulatedas a depot preparation. Such a long acting depot formulation can beadministered by implantation, for example subcutaneously orintramuscularly; or by intramuscular injection. Thus, for example, thecompounds can be formulated with suitable polymeric or hydrophobicmaterials, for example as an emulsion in an acceptable oil; or ionexchange resins; or as a sparingly soluble derivatives, for example, asa sparingly soluble salt.

A therapeutically effective amount of a compound of the invention may beadministered as a single pulse dose, as a bolus dose, or as pulse dosesadministered over time. Thus, in pulse doses, a bolus administration ofa compound of the invention is provided, followed by a time periodwherein no a compound of the invention is administered to the subject,followed by a second bolus administration. In specific, non-limitingexamples, pulse doses of a compound of the invention are administeredduring the course of a day, during the course of a week, or during thecourse of a month.

In one embodiment, a therapeutically effective amount of a compound ofthe invention is administered with a therapeutically effective amount ofanother agent, for example a further anti-neoplastic chemotherapeuticagent (for example, thalidomide, dexamethasone, bortezomib,lenalidomide, melphalan, cisplatinum, doxorubicin, 5-FU, etc) or anagent to treat anaemia (for example erythropoietin), or an agent toprevent bone fractures (for example a bisphosphonate such as pamidronateor zoledronic acid).

The therapeutically effective amount of a compound of the invention willbe dependent on the molecule utilized, the subject being treated, theseverity and type of the affliction, and the manner and route ofadministration.

According to the third aspect of the invention, there is provided amethod of treating a disorder or disease comprising administering acompound according to the first and second aspect of the invention or apharmaceutical composition according to the second aspect of theinvention administering a therapeutically effective amount of a compoundaccording to the first aspect of the invention or a pharmaceuticalcomposition according to the second aspect of the invention to a subjectin need thereof.

Disorders and Diseases

The compounds, compositions and methods of the invention are suitablefor the treatment or prevention of diseases and disorders which areeither characterised by aberrant increased expression or activity ofGadd45β or which are characterised by aberrant activation of the NF-κBpathway and are amenable to treatment by the induction of ProgrammedCell Death by the inhibition of Gadd45β activity.

Diseases suitable for treatment or prevention include cancer. Preferablythe cancer is a cancer expressing raised levels of Gadd45β relative tocorresponding normal healthy cells or tissues. Cancers known to expressaberrantly high levels of Gadd45β and so suitable for treatment with thecompounds of the invention include: multiple myeloma, diffuse largeB-cell lymphoma, Burkitt's lymphoma, promonocytic leukaemia and otherleukemias, as well as solid tumours such as hepatocellular carcinoma,bladder cancer, brain and central nervous system cancer, breast cancer,head and neck cancer, lung cancer, and prostate cancer. According tocertain embodiments the cancer is a cancer that depends on NF-κB for itssurvival. Specific such cancers that depend on NF-κB for survival and soare suitable for treatment or prevention include: multiple myeloma,mantle cell lymphoma, MALT lymphoma, Hodgkin's lymphoma, diffuse largeB-cell lymphoma, Burkitt's lymphoma, promonocytic leukaemia,myelodysplastic syndrome, adult T-cell leukaemia (HTLV-1), chroniclymphocytic leukaemia, chronic myelogenous leukemia, acute myelogenicleukaemia, acute lymphoblastic leukemia, colitis-associated cancer,colon cancer, liver cancer (for example hapatocellular carcinoma)cervical cancer, renal cancer, lung cancer, oesophageal cancer, gastriccancer, laryngeal cancer, prostate cancer, pancreatic cancer, thyroidcancer, parathyroid cancer, bladder cancer, ovarian cancer, breastcancer, melanoma, cylindroma, squamous cell carcinoma (skin, and headand neck), oral carcinoma, endometrial carcinoma, retinoblastoma,astrocytoma, and glioblastoma. According to certain preferredembodiments the cancer is multiple myeloma. According to certainembodiments, cells taken from the subject (for example biopsied from asubject's cancer or extracted from the subjects blood or other bodyfuild into which they may have been released by the cancer) may betested for NF-κB activation and/or elevated level of Gadd45β activity inorder to determine the cancer's suitability to treatment by methods,compounds and compositions of the invention.

Other diseases and disorders suitable for treatment or preventioninclude autoimmune disease (for example multiple sclerosis, lupus,type-I diabetes), allergic disease (for example asthma), chronicinflammatory disease (for example inflammatory bowel disease, rheumatoidarthritis, psoriasis, ulcerative colitis), genetic disease (for example,incontinentia pigmenti, anhidrotic ectodermal dysplasia withimmunodeficiency and cylindromatosis), ischemic and vascular disease(for example atherosclerosis, angina pectoris, stroke, myocardialinfarction), and degenerative disease (for example Alzheimer's andParkinson disease), liver diseases such as liver fibrosis and livercirrhosis

A broad range of diseases and disorders depend on the activity of NF-κB.Indeed, the pathogenesis of virtually every known human disease ordisorder is now being considered to depend on inflammation, and hence toinvolve NF-κB. This functions as a masterswitch of the inflammatoryresponse, coordinating expression of an array of over 200 genes encodingcytokines, receptors, transcription factors, chemokines,pro-inflammatory enzymes, and other factors, including pro-survivalfactors, which initiate and sustain inflammation. The compounds of theinvention inhibit the discrete pro-survival activity of NF-κB ininflammation. Therefore, diseases and disorders amenable to treatmentwith these compounds include, apart from conventional chronicinflammatory diseases (such as inflammatory bowel disease, rheumatoidarthritis, and psoriasis), other diseases and disorders that depend on asignificant inflammatory component. Examples of such diseases anddisorders, which are being treated with anti-inflammatory agents orNF-κB-inhibiting agents or have been proposed as suitable for treatmentwith NF-κB inhibitors and could also be treated with a compound of theinvention, include:

1. respiratory tract: obstructive diseases of the airways including:asthma, including bronchial, allergic, intrinsic, extrinsic,exercise-induced, drug-induced (including aspirin and NSAID-induced) anddust-induced asthma, both intermittent and persistent and of allseverities, and other causes of airway hyper-responsiveness; chronicobstructive pulmonary disease (COPD); bronchitis, including infectiousand eosinophilic bronchitis; emphysema; bronchiectasis; cystic fibrosis;sarcoidosis; farmer's lung and related diseases; hypersensitivitypneumonitis; lung fibrosis, including cryptogenic fibrosing alveolitis,idiopathic interstitial pneumonias, fibrosis complicatinganti-neoplastic therapy and chronic infection, including tuberculosisand aspergillosis and other fungal infections; complications of lungtransplantation; vasculitic and thrombotic disorders of the lungvasculature, and pulmonary hypertension; antitussive activity includingtreatment of chronic cough associated with inflammatory and secretoryconditions of the airways, and iatrogenic cough; acute and chronicrhinitis including rhinitis medicamentosa, and vasomotor rhinitis;perennial and seasonal allergic rhinitis including rhinitis nervosa (hayfever); nasal polyposis; acute viral infection including the commoncold, and infection due to respiratory syncytial virus, influenza,coronavirus (including SARS) or adenovirus; or eosinophilic esophagitis;2. bone and joints: arthritides associated with or includingosteoarthritis/osteoarthrosis, both primary and secondary to, forexample, congenital hip dysplasia; cervical and lumbar spondylitis, andlow back and neck pain; osteoporosis; rheumatoid arthritis and Still'sdisease; seronegative spondyloarthropathies including ankylosingspondylitis, psoriatic arthritis, reactive arthritis andundifferentiated spondarthropathy; septic arthritis and otherinfection-related arthopathies and bone disorders such as tuberculosis,including Potts' disease and Poncet's syndrome; acute and chroniccrystal-induced synovitis including urate gout, calcium pyrophosphatedeposition disease, and calcium apatite related tendon, bursal andsynovial inflammation; Behcet's disease; primary and secondary Sjogren'ssyndrome; systemic sclerosis and limited scleroderma; systemic lupuserythematosus, mixed connective tissue disease, and undifferentiatedconnective tissue disease; inflammatory myopathies includingdermatomyositits and polymyositis; polymalgia rheumatica; juvenilearthritis including idiopathic inflammatory arthritides of whateverjoint distribution and associated syndromes, and rheumatic fever and itssystemic complications; vasculitides including giant cell arteritis,Takayasu's arteritis, Churg-Strauss syndrome, polyarteritis nodosa,microscopic polyarteritis, and vasculitides associated with viralinfection, hypersensitivity reactions, cryoglobulins, and paraproteins;low back pain; Familial Mediterranean fever, Muckle-Wells syndrome, andFamilial Hibernian Fever, Kikuchi disease; drug-induced arthalgias,tendonititides, and myopathies;3. pain and connective tissue remodelling of musculoskeletal disordersdue to injury [for example sports injury] or disease: arthitides (forexample rheumatoid arthritis, osteoarthritis, gout or crystalarthropathy), other joint disease (such as intervertebral discdegeneration or temporomandibular joint degeneration), bone remodellingdisease (such as osteoporosis, Paget's disease or osteonecrosis),polychondritits, scleroderma, mixed connective tissue disorder,spondyloarthropathies or periodontal disease (such as periodontitis);4. skin: psoriasis, atopic dermatitis, contact dermatitis or othereczematous dermatoses, and delayed-type hypersensitivity reactions;phyto- and photodermatitis; seborrhoeic dermatitis, dermatitisherpetiformis, lichen planus, lichen sclerosus et atrophica, pyodermagangrenosum, skin sarcoid, discoid lupus erythematosus, pemphigus,pemphigoid, epidermolysis bullosa, urticaria, angioedema, vasculitides,toxic erythemas, cutaneous eosinophilias, alopecia greata, male-patternbaldness, Sweet's syndrome, Weber-Christian syndrome, erythemamultiforme; cellulitis, both infective and non-infective; panniculitis;cutaneous lymphomas, non-melanoma skin cancer and other dysplasticlesions; drug-induced disorders including fixed drug eruptions;5. eyes: blepharitis; conjunctivitis, including perennial and vernalallergic conjunctivitis; iritis; anterior and posterior uveitis;choroiditis; autoimmune; degenerative or inflammatory disordersaffecting the retina; ophthalmitis including sympathetic ophthalmitis;sarcoidosis; infections including viral, fungal, and bacterial;6. gastrointestinal tract: glossitis, gingivitis, periodontitis;oesophagitis, including reflux; eosinophilic gastro-enteritis,mastocytosis, Crohn's disease, colitis including ulcerative colitis,proctitis, pruritis ani; coeliac disease, irritable bowel syndrome, andfood-related allergies which may have effects remote from the gut (forexample migraine, rhinitis or eczema);7. abdominal: hepatitis, including autoimmune, alcoholic and viral;fibrosis and cirrhosis of the liver; cholecystitis; pancreatitis, bothacute and chronic;8. genitourinary: nephritis including interstitial andglomerulonephritis; nephrotic syndrome; cystitis including acute andchronic (interstitial) cystitis and Hunner's ulcer; acute and chronicurethritis, prostatitis, epididymitis, oophoritis and salpingitis;vulvo-vaginitis; Peyronie's disease; erectile dysfunction (both male andfemale);9. allograft rejection: acute and chronic following, for example,transplantation of kidney, heart, liver, lung, bone marrow, skin orcornea or following blood transfusion; or chronic graft versus hostdisease;10. CNS: Atzheimer's disease and other dementing disorders including CJDand nvCJD; amyloidosis; multiple sclerosis and other demyelinatingsyndromes; cerebral atherosclerosis and vasculitis; temporal arteritis;myasthenia gravis; acute and chronic pain (acute, intermittent orpersistent, whether of central or peripheral origin) including visceralpain, headache, migraine, trigeminal neuralgia, atypical facial pain,joint and bone pain, pain arising from cancer and tumor invasion,neuropathic pain syndromes including diabetic, post-herpetic, andHIV-associated neuropathies; neurosarcoidosis; central and peripheralnervous system complications of malignant, infectious or autoimmuneprocesses;11. other auto-immune and allergic disorders including Hashimoto'sthyroiditis, Graves' disease, Addison's disease, diabetes mellitus,idiopathic thrombocytopaenic purpura, eosinophilic fasciitis, hyper-IgEsyndrome, antiphospholipid syndrome;12. other disorders with an inflammatory or immunological component;including acquired immune deficiency syndrome (AIDS), leprosy, Sezarysyndrome, and paraneoplastic syndromes;13. cardiovascular: atherosclerosis, affecting the coronary andperipheral circulation; pericarditis; myocarditis, inflammatory andauto-immune cardiomyopathies including myocardial sarcoid; ischaemicreperfusion injuries; endocarditis, valvulitis, and aortitis includinginfective (for example syphilitic); vasculitides; disorders of theproximal and peripheral veins including phlebitis and thrombosis,including deep vein thrombosis and complications of varicose veins;14. gastrointestinal tract: Coeliac disease, proctitis, eosinopilicgastro-enteritis, mastocytosis, Crohn's disease, ulcerative colitis,microscopic colitis, indeterminant colitis, irritable bowel disorder,irritable bowel syndrome, non-inflammatory diarrhea, food-relatedallergies which have effects remote from the gut, e.g., migraine,rhinitis and eczema.

According to a forth aspect of the invention, there is provided acompound according to the first aspect of the invention or a compositionaccording to the second aspect of the invention for use as a medicament.

According to a fifth aspect of the invention, there is provided use of acompound according to the first aspect of the invention or apharmaceutical composition according to the second aspect of theinvention for the manufacture of a medicament for the treatment of adisease or disorder. Said disease or disorder and subject being definedin certain preferred embodiments as described above in reference to thethird aspect of the invention.

Preferably products, methods of the invention are for the treatment ofdiseases and disorders in humans.

Theranostic Aspects of the Invention

The invention encompasses in various embodiments methods of treatment,use of compounds or compositions of the invention of the manufacture ofa medicament and compounds or compositions of the invention fortherapeutic use.

According to certain embodiments the invention may also encompass:

a) Methods of treating or preventing a disease or disorder as statedabove wherein the disease or disorder is a cancer in an individualsubject and that subject's suspected cancer has been previously sampled(for example by taking a tissue biopsy or body fluid such as blood orsputum) and determined to show elevated levels of Gadd45β expressionand/or activity and/or elevated levels of NF-κB expression and/oractivity.b) Compounds or compositions of the invention for use as a medicamentfor treatment of tissues of an individual previously determined to showelevated levels of Gadd45β expression and/or activity and/or elevatedlevels of NF-κB expression and/or activity.c) Use of compounds or compositions of the invention for the manufactureof a medicament for the treatment of a disease or disorder which iseither characterised by aberrant increased expression and/or activity ofGadd45β or which are characterised by aberrant activation of the NF-κBpathway and are amenable to treatment by the induction of ProgrammedCell Death by the inhibition of Gadd45β activity wherein the disease ordisorder is a cancer wherein said cancer cells have previously beendetermined to show elevated levels of Gadd45β expression and/oractivity.

“Elevated levels” may mean elevated by at least 10%, at least 20%, atleast 30%, at least 50%, at least 75%, at least 100%, at least 200%, atleast 300%, at least 400%, at least 500%, at least 600%, at least 700%,at least 800%, at least 900%, or at least 1,000% compared to levels incontrol healthy tissue of the same origin and optionally obtained fromthe same subject or from a healthy subject. Levels of expression andactivity may be determined by any method known in the art includingRT-PCR, Southern blotting, Northern blotting, Western blotting, ELISA,radio-immuno assay, kinase assay and other binding, functional, and/orexpression assays.

This theranostic aspect of the invention is primarily illustrated by theresults presented in FIGS. 12A and 12B. The results shown heredemonstrate that, in a panel of 29 cancer cell lines of differenttissues of origin, cancer cell sensitivity to Z-DTP-induced killingcorrelates with a very high degree of statistical significance withlevels of endogenus Gadd45β expression, as assessed by qRT-PCR assays.Indeed, the correlation plot of Gadd45β expression versus the percentageof cell survival/proliferation after treatment with Z-DTP2 shows thatthe significance of the correlation coefficient between the 2parameters' domain is very high (p<0.01) (Pearson correlation).Strikingly, the only multiple myeloma cell line (out of a total of 9multiple myeloma cell lines tested) which is refractory toZ-/mDTP-induced killing, as well as to cell death induced by thesh-RNA-mediated silencing of Gadd45β (FIGS. 16, 17, and 18), is theRPMI-8226 cell line, which expresses the lowest—almostundetectable—levels of Gadd45β (FIG. 12A). These data indicate thatshould DTP-based therapy enter the clinic, it will be possible topredict patient responder populations via simple and cost-effectiveqRT-PCR analysis. For example, primary cell from multiple myelomapatients can be analyzed for levels of Gadd45β expression, and patientswith high levels of this expression can be deemed as those who willreceive the most benefit from treatment with the compounds of theinvention. Hence, an important aspect of the invention is a theranosticaspect—that is the application of a clinically useful assay to predictDTPs' therapy response in patients.

This theranostic aspect of the invention is also supported by the veryhigh target specificity of the compounds of the invention in cells forthe Gadd45β/MKK7 complex. This indicates that the higher the levels ofexpression of the target (i.e. Gadd45β) in cells, the higher will be theprobability that such cells will depend on Gadd45β for survival, hencethe higher will be the probability that such cells will be sensitive toZ-/mDTP-induced killing. This high specificity of Z-/mDTPs isdemonstrated by the findings that: 1) In a large panel of tumour celllines there is a highly significant statistical correlation betweenlevels of Gadd45β expression and cancer cell sensitivity toZ-/mDTP-induced killing (FIG. 12); 2) the sh-RNA-mediated downregulationof Gadd45β rapidly induces apoptosis in Z-/mDTP-sensitive but not inZ-/mDTP-resistant cancer cell lines (FIGS. 16, 17, 18), and the kineticsof apoptosis induction by Gadd45β-specific sh-RNAs in these cell linesis similar to those observed with Z-/mDTPs (FIGS. 7A, 8B, and 8C); 3)the sh-RNA-mediated downregulation of MKK7 renders Z-/mDTP-sensitivecancer cell lines completely resistant to Z-/mDTP-induced killing (FIGS.20A, 20B, and 20C); 4) the therapeutic target of the invention is theinterface between two proteins, Gadd45β and MKK7 (FIGS. 21A, 21B, 21C,and 21D)—which further provides potential for high target selectivity, akey advantage of our solution over existing therapies. These data,together with the low toxicity of Z-/mDTPs to normal cells and thefindings that knockout ablation of Gadd45β is well tolerated in mice(see reference by Papa, et al. (2008) J. Clin. Invest. 118:191-1923),indicate that targeting the discreet pro-survival functions of NF-κB incell survival via Z-/mDTP-mediated inhibition of Gadd45β/MKK7 canprovide a therapy that is more specific, less toxic, and hence moreeffective than therapies targeting the NF-κB pathway and/or theproteasome.

EXAMPLES

The following non-limiting examples illustrate the invention.

Example 1 Synthesis of Z-DTP2

By way of example, the synthesis of Z-DTP2 is reported. Z-DTP2 comprisesa tetrapeptide core made up of D-tyrosine, D-glutamine, D-arginine,D-phenylalanine with benzyloxycarbonyl (that is a Z group) bonded to theN-terminus by means of an amide bond and an amino group bonded to theC-terminus by means of an amide bond.

Materials and Methods

Z-DTP2 was manually prepared following the Fmoc/tBu solid phase method(Fields G. B. and Noble R. L. 1990 Int J Pept Protein Res; 35: 161-214;Bodansky, M. and Bodansky A. 1995). The practice of peptide synthesis,2nd edn., Springer Verlag, Berlin) and starting from 500 μmoles (1000mg) of Rink amide polystyrene resin (Fmoc-RINK-AM-resin, GL Biochem,Shangai, China, Cat. 49001), having a substitution of 0.50 mmoles/g. Theresin was placed in a 30 mL polypropylene vessel endowed with a 20 μmteflon septum, a polypropylene upper cap and a lower luer-lockpolypropylene cap. The resin was swollen with 10.0 mL of a 50:50dichloromethane (DCM):dimethyl formamide (DMF) mixture (both fromLabScan, Stillorgan, Ireland; DCM cat. No H6508L; DMF cat. No H33H11X)for 20 minutes. Then after solvent removal under vacuum, the Fmoc groupwas cleaved by treatment with 5.0 mL of a DMF-Piperidine 8:2 mixture(Piperidine, Pip, cat. No Cat. No 80641; Sigma-Aldrich, Milan, Italy)for 20 minutes at room temperature (RT). The reactant was removed undervacuum and the resin washed 3 times with 5.0 mL of DMF. Then, 2.5mmoles, 0.97 g, of Fmoc-D-Phe-OH (GL Biochem, Shangai. Cat. N. 35702)were dissolved in 5.0 mL of DMF (final conc. 0.5 M) and activated with5.0 mL of a 0.5 M solution ofBenzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate(PyBOP, Novabiochem, cat. No 01-62-0016) in DCM, and 0.90 mL ofdi-iso-propyl-ethylamine (5.0 mmoles; DIEA, Sigma-Aldrich, cat. NoD-3887). The solution of activated aminoacid was poured onto the resinand left under vigorous stirring for 30 minutes. The solution wasdrained under vacuum and the resin washed 3 times with 5.0 mL of DMF.The Fmoc group on the α-NH₂ was removed as described earlier using a 8:2DMF-Pip solution (5.0 mL) for 20 minutes and extensive washing with 5.0mL of DMF (3 times). A solution of Fmoc-D-Arg(Pbf)-OH (2.5 mmoles, 1.6 gin 5.0 mL DMF; GL Biochem, Shangai, Cat. N. 36404) was activated asdescribed using 2.5 mmoles of PyBOP and 5.0 mmoles of pure DIEA. Thesolution was transferred onto the resin and left under stirring for 30minutes. After cleavage of the Fmoc groups with 5.0 mL of a 8:2 DMF-Pipsolution and washing with DMF (3 times, 5.0 mL), a solution ofFmoc-(D)-Glu(tBu)-OH 0.50 M in DMF (2.5 mmoles, 1.1 g in 5.0 mL DMF; GLBiochem, Shangai, Cat. N. 36605) preactivated with PyBOP and DIEA asdescribed above, was added to the resin and the reaction was left toproceed for 30 minutes at room temperature. Following draining of theaminoacid, the Fmoc-group was removed as described above (20 minutetreatment with 8:2 DMF:Pip, 5.0 mL) and the resin washed 3 times with5.0 mL of DMF. 2.5 mmoles of Fmoc-(D)-Tyr(tBu)-OH (1.2 g, GL Biochem,Shangai, Cat. N. 36906) dissolved in 5.0 mL of DMF was preactivated withPyBOP and DIEA as reported above, was transferred onto the resin andleft under stirring for 45 minutes. The aminoacid solution was removedby vacuum draining, then the resin was washed 5 times with 5.0 mL ofDMF. 5 mmoles of Z-OSu (benzyloxycarbonyl-N-hydroxy-succinimide, GLBiochem, Shangai, Cat. N. 10502) were dissolved in 10 mL of DMF andadded to the resin. 2.4 mL of DIEA were added and the reaction was leftunder stirring over night. After draining of the solution, the resin wasextensively washed with DMF, DCM, methyl alcohol (MeOH, LabScan, Cat. NoC2517), and ethyl ether (Et₂O, LabScan, Cat. No A3509E), and dried undervacuum and weighted. The weight was 1.1 g. To cleave the peptide, theresin was treated with 10.0 mL of a mixture composed of TFA-H₂O-TIS90:5:5 (v/v/v) mixture (TFA, trifluoroacetic acid, Sigma-Aldrich, ItalyCat. No 91700; TIS, tri-iso-propylsilane, Sigma-Aldrich, cat. N.23,378-1) for 3 hours at RT. The resin was removed by filtration, then20 mL of cold Et₂O was added to the trifluoroacetic solution, leading tothe formation of a white precipitate. After removal of the solvents bycentrifugation, the precipitate was washed with 10.0 mL of cold Et₂O,dissolved in 10.0 mL of H₂O/CH₃CN 50:50 (v/v) and lyophilized. Thepeptide was characterized by LC-MS using a narrow bore 50×2 mm ID ONYXC18 column (Phenomenex, Torrance, Calif., USA), equilibrated at 600μL/min with 5% CH₃CN, 0.05% TFA. The analysis was carried out applying agradient of CH₃CN, 0.05% TFA from 5% to 70% over 3 minutes. The peptidewas purified by semi-preparative RP-HPLC using a 10×1 cm C18 ONYX column(Phenomenex, Torrance, Calif., USA), equilibrated at 20 mL/min,injecting 20 mg in each run. A gradient from 5% to 65% over 8 minuteswas applied to elute the peptide. Pure fractions were pooled andcharacterized by LC-MS. The determined MW of Peptide A was 746.8 amu(theor. 746.83 amu) and the product was more than 95% pure (HPLC). Ayield of around 60% was achieved after purification of all the crudeproduct.

Example 2 Dose Dependent Inhibition of the Interaction Between Gadd45βand MKK7 with a Selection of Compounds of General Formula (I)

To evaluate the inhibitory properties of peptides, ELISA-based assayswere performed. In these assays, a fusion protein of glutathioneS-transferase (GST) and mitogen-activated protein kinase kinase 7 (MKK7)was coated onto wells of a 96-well plate, while biotinylated-hGadd45βwas used in solution. hGadd45β was biotinylated using an EZ LinkNHS-LC-biotin kit (Pierce, Rockford, Ill.), according to Tornatore etal. (Tornatore L., et al. (2008). J Mol Biol; 378:97-111).

Materials and Methods

Firstly, the association between Gadd45β and MKK7 was investigated byELISA assays as also reported in Tornatore et al. (Tornatore L., et al.(2008). J Mol Biol; 378:97-111). The GST-fused full-length kinase wascoated for 16 h at 4° C., at a concentration of 42 nM in buffer A (25 mMTris pH 7.5, 150 mM NaCl, 1 mM DTT and 1 mM EDTA) into wells of a96-well microtiter plate. Some wells were filled with buffer alone andwere used as blanks After incubation for 16 h at 4° C., the solutionswere removed and the wells were filled with 350 μL of a 1% (w/v)solution of NFDM (Non Fat Dry Milk) in PBS (phosphate buffered saline).The plate was incubated for 1 h at 37° C. in the dark. After washingwith buffer T-PBS (PBS with 0.004% (v/v) Tween detergent), the wellswere filled with 100 μL of biotinylated-hGadd45β at concentrationsranging from 8.4 nM to 168 nM. Each data point was performed intriplicate. Following incubation for 1 hr in the dark at 37° C. thesolutions were removed and the wells were again washed with T-PBS. Then100 μL of a 1:10,000 dilution of horseradish peroxidase-conjugatedstreptavidin dissolved in buffer was added to each well and the plateincubated for 1 hr at 37° C. in the dark. After removal of the enzymesolution and washing, 100 μL of the chromogenic substrateo-phenylendiamine (0.4 mg/mL in 50 mM sodium phosphate-citrate buffer,containing 0.4 mg/mL of urea in hydrogen peroxide) was added and thecolour was allowed to develop in the dark for 5 min. The reaction wasstopped by adding 50 μL of 2.5 M H₂SO₄. The absorbance at 490 nm wasmeasured in all wells and the values were averaged after subtracting thecorresponding blanks Bound protein was then detected as described above.The molar concentration of biotinylated-hGadd45β at which thehalf-maximal ELISA signal is detected corresponds to the dissociationconstant (K_(D)) (Friguet B, Chaffotte A F, Djavadi-Ohaniance L,Goldberg M E. J Immunol Methods. 1985 Mar. 18; 77(2):305-19). Bindingcompetition assays were performed by coating GST-MKK7 at 42 nM asdescribed, a concentration of biotinylated-hGadd45β of 21 nM(pre-saturation conditions 1:0.5 mol/mol ratio) and, in a first test,using competitors at 21 nM. The binding of biotinylated hGadd45β toGST-MKK7 was analyzed in the presence of increasing amounts ofcompetitor peptide (concentrations ranging from 0.01 nM to 100 nM), andthe values obtained with the competitor were expressed as the percentageof the binding detected in the absence of competitor. Data of activity,expressed as percentage of inhibiting capacity at 21 nM under the assayconditions, are reported in the following Table I for a selected set ofcompounds according to the invention. According to the conventionadopted in the table “L-Xaa” and “D-Xaa” refer to the L and D forms ofamino acid Xaa.

Data of IC₅₀ of selected compounds (i.e. the compound dose required toachieve a 50% reduction of Gadd45β binding to MKK7) are reported in FIG.3C.

Example 3 Isolation of Lead Tetrapeptides

Materials and Methods

An ELISA screen was used to identify lead D-tetrapeptides from whichpreferred compounds of the invention could be derived. A simplifiedcombinatorial peptide library (Marasco et al. 2008, Curr. Protein Pept.Sci. 9:447-67) was screened for antagonists of the Gadd45β/MKK7interaction. This library contained a total of 12⁴=20,736 differenttetrapeptides formed by combinations of the following amino acidresidues Gln (Q), Ser (S), Arg (R), Ala (A), Tyr (Y), Pro (P), Met (M),Cys (C), Phe (F), Leu (L), His (H), Asp (D), and was iterativelydeconvoluted in four steps by ELISA competition assays using at eachstep coated MKK7 (42 nM), soluble-biotin-hGadd45β (21 nM) and each ofthe 12 sub-libraries (42 nM). The results of this screen are shown inTable I above (wherein standard single letter amino acid residue codesare used and X₂, X₃ and X₄ represent mixtures of the 12 residues givenabove) (see also FIG. 3A). The resulting most active peptide describedin Table I (i.e. Fmoc-(βAla)₂-YDHF-NH₂, also referred to as Fmoc-LTP1)was then subjected to several rounds of optimization and removal of theFmoc-(βAla)₂-tag, yielding Ac-LTP1 and Ac-LTP2 (see Table II). Thesetetrapeptides were then resynthesized using D-isomers of the same aminoacids, ultimately yielding the lead tetrapeptide 1 and 2 (DTP1 andDTP2), which disrupted the Gadd45β/MKK7 interaction with IC₅₀s of 0.22nM and 0.19 nM, respectively (FIG. 3C).

Sequence of DTP1: [SEQ ID NO.: 38]Acetyl-(D-Tyr)-(D-Asp)-(D-His)-(D-Phe)-NH₂ Sequence of DTP2:[SEQ ID NO.: 37] Acetyl-(D-Tyr)-(D-Glu)-(D-Arg)-(D-Phe)-NH₂

Also the following sequences were selected as negative controls (NC):

Sequence of NC1: Acetyl-(D-Tyr)-(D-Asp)-(D-His)-(D-Gln)-NH₂[SEQ ID NO.:81]

Sequence of NC2: Acetyl-(L-Tyr)-(L-Asp)-(L-His)-(L-Ala)-NH₂ [SEQ ID NO.:82]

Sequence of NC3: [SEQ ID NO.: 83]Acetyl-(L-Tyr)-(L-Glu)-(L-Lys)-(L-Trp)-NH₂ Sequence of NC4:[SEQ ID NO.: 84] Acetyl-(L-Tyr)-(L-Asp)-(L-Lys)-(L-Trp)-NH₂

FIGS. 3A, 3B and 3C show the ELISA competition binding assays.Percentage inhibition of acetylated peptides and/or modified peptides(that is peptides conjugated to either acetyl or other groups) are shownrespectively in Tables II and III (which use standard single letteramino acid residue codes).

Example 4 Immunoprecipitation Assays

Materials and Methods

Human Embryonic Kidney (HEK-293) were cultured in Dulbecco's modifiedEagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS), 100units/ml penicillin, 100 mg/mL streptomycin, and 1% glutamine. HEK-293cells (2.2×10⁶) were seeded onto 10 cm² tissue-culture dishes, and thefollowing day, were transfected with pcDNA-FLAG-MKK7 andpcDNA-HA-Gadd45β plasmids, using a standard Ca₃(PO4)₂ precipitationtechnique (Papa, S et al., (2004) Nat. Cell Biol. 6, 146-153).Forty-eight hours after transfection, the cells were washed once withPBS, then resuspended and incubated for 30 min at 4° C. in lysis buffer(20 mM HEPES, 350 mM NaCl, 20% glycerol, 1 mM MgCl₂, 0.2 mM EGTA, 1 mMDTT, 1 mM Na₃VO₄, and 50 mM NaF) supplemented with protease inhibitors(1 mM phenylmethylsulfonylfluoride, 10 μM chymostatin, 2 μg/mlaprotinin, and 2 μg/ml leupeptin) with occasional gentle shaking. Thelysed cells were collected and then centrifuged at 45,000×g for 40 min.The resulting cleared cell lysates were used for further analysis.

Lead tetrapeptides DTP1 and DTP2 isolated in Example 3, together withnegative control tetrapeptides (NC1, NC2, NC3 and NC4), wereco-incubated with Gadd45β/MKK7 in order to demonstrate that the activeD-tetrapeptides, but not the negative control tetrapeptides, disruptedthe Gadd45β/MKK7 interaction. Immunoprecipitations were performed usingessentially the same conditions described in Papa, S et al., (2004) Nat.Cell Biol. 6, 146-153 and the references therein, and an anti-FLAGantibody which precipitated FLAG-tagged MKK7. Western blots weredeveloped using anti-MKK7 antibodies or anti-HA antibodies (binding toHA-hGadd45β), as indicated in FIG. 5 (bottom and top panels,respectively).

Results

Results are presented in FIG. 5. It can be seen from the western blotspresented in FIG. 5 that there was a strong interaction between Gadd45βand MKK7 when co-immunoprecipitation was performed with lysates fromHEK-293 cells transiently expressing HA-Gadd45β and FLAG-MKK7 and ananti-FLAG antibody (specifically binding to FLAG-tagged MKK7). Thisresult was obtained when co-immunoprecipitations were performed eitherin the absence of tetrapeptides or in the presence of negative control(NC) D-tetrapeptides NC1, NC2, NC2 or NC4. When co-immunoprecipitationswere performed in the presence of 1 or 5 nM of DTP1 or DTP2, however,the precipitated complex contained no or very little Gadd45β, indicatingthat the interaction between MKK7 and Gadd45β had been disrupted by theactive DTP compounds, thereby leading to a reduction of Gadd45β in theco-immuno-precipitates. These data confirm and extend the resultobserved in the ELISA competition assays shown in FIGS. 3A, 3B and 3Cand FIG. 4.

Example 5 Stability of DTPs in Human Serum

Materials and Methods

In FIG. 4, Gadd45β/MKK7 binding, competition ELISA assays were carriedout to determine the stability of Z-conjugated D-tetrapeptides in humanserum. For this purpose, the activities of the most active Gadd45β/MKK7antagonists selected from the combinatorial library screen described inExample 3 (i.e. Z-LTP1, Z-LTP2, Z-DTP1, and Z-DTP2), as well as of onenegative control L-tetrapeptide (i.e. Z-LNC) and the correspondingD-enantiomer (i.e. Z-DNC), were compared before and after a 48-hrpre-incubation with human serum at 37° C. in ELISA competition assays.ELISA were performed as described in FIG. 3 C. Briefly, 100 μl of 42 nMof recombinant GST-MKK7 in ELISA buffer (25 mM Tris pH 7.5, 150 mM NaCl,1 mM DTT, 1 mM EDTA) were coated onto wells of 96-well plates byovernight incubation at 4° C. After blocking with 2% of NFDM for 1 hr at37° C., plates were washed with TPBS, and then 21 nM of recombinant,biotinylated human (h)Gadd450 were added to the wells together withincreasing concentrations of tetrapeptides which had either beensubjected or had not been subjected to pre-incubation with human serum,as indicated. For a further discussion of the conditions used for thecompetition ELISA assay, the reader is directed to Tornatore et al 2008JMB, 378: 97-111 and the references therein.

Results

FIG. 3C shows that the activities of DTP1 and DTP2 are comparable tothose of their corresponding L-enantiomers (i.e. LTP1 and LTP2,respectively) in inhibiting the formation of the Gadd45β/MMK7 complex,as shown by a comparison the IC₅₀s of DTPs and LTPs in ELISA competitionassays. FIG. 4 shows that no loss of activity occurs after a 48-hrincubation of Z-DTP1 or Z-DTP2 with human serum at 37° C. Indeed, thedata show that after this pre-incubation, Z-DTPs but not Z-LTPs fullyretain their ability to disrupt the Gadd45β-MKK7 interaction in ELISAcompetition assays. By comparing the IC₅₀s of the tetrapeptides afterpre-incubation with serum and after no pre-incubation with serum, it canbe seen that Z-DTP1 and Z-DTP2 are completely stable afterpre-incubation with serum (IC₅₀=0.19 nM for Z-DTP1, and IC₅₀=0.18 nM forZ-DTP2), whereas Z-LTP1 and Z-LTP2 are not, as the latter tetrapeptidesshow significant loss of activity after pre-incubation with serum (seetheir IC₅₀s>10 μM). At all the concentrations tested, the inhibitoryactivities of the D-tetrapeptides that had been pre-incubated with serumwere indistinguishable in these assays from those of the D-tetrapeptidesthat had not been subjected to this pre-incubation (FIGS. 3C and 4).

The comparison of the dose-dependent patterns shown in FIGS. 3C and 4indicates that Z-DTP1 and Z-DTP2 are stable in human serum at 37° C. andso are suitable for systemic use, whereas Z-LTP1 and Z-LTP2 are not. Itcan also be seen that negative control tetrapeptides (e.g. Z-DNC andZ-LNC) lack any activity in the aforementioned competition ELISA assays,regardless of whether or not they had been pre-incubated with humanserum (FIGS. 3C and 4). The data depicted in FIGS. 3C and 4 also showthat the N-terminal addition of a benzyloxycarbonil (Z) group (in placeof the acetyl group) does not compromises the ability of either DTP1 andDTP2 or of LTP1 and LTP2 to inhibit formation of the Gadd45β/MKK7complex—yet the addition of a Z group markedly increases DTPs' cellularuptake (data not shown), hence markedly increases DTPs' cellularactivity in tumour killing assays (see FIGS. 7A and 7B).

Example 6 Determination of the IC₅₀s of Z-DTPs in a Panel of MultipleMyeloma Cell Lines

Materials and Methods

To further examine the effects of D-tetrapetide treatment on thesurvival/proliferation of multiple myeloma cell lines, the cells fromthe 8 multiple myeloma cell lines (out of the 9 multiple myeloma celllines tested) that were sensitive to Z-DTP-induced killing (i.e. U266,KMS-11, NC1-H929, ARH-77, JJN-3, KMS-12, KMS-18, KMS-27 cells; see alsoFIGS. 8A, 8B, 8C, and 12) were treated with increasing concentrations(ranging from 0.01 to 10 μM) of Z-DTP1 or Z-DTP2 for 24, 72 or 144 hrs,as shown in Table IV. Cultures of multiple myeloma cells and treatmentswith Z-DTPs were carried out as described in Example 8 (see below). Theeffects of Z-DTPs on the survival/proliferation of multiple myelomacells were evaluated by the use of [³H]thymidine) incorporation assays,performed as also described in Example 8. The amount of cellproliferation measured with each of the Z-DTPs' concentrations used andwith the untreated cultures (i.e. cultures incubated with medium alone),was expressed as counts per minute (c.p.m.)—which directly correlatewith the extent of cell proliferation. All experiments were performed intriplicate. The mean concentrations of Z-DTP1 and Z-DTP2, as well as oftheir derivatives (e.g. mDTP3), that resulted in 50% inhibition of cellproliferation (i.e. IC₅₀) relative to the cell proliferation recordedwith the untreated cultures were then determined. The IC₅₀s of Z-DTP1and Z-DTP2 calculated for the 8 sensitive multiple myeloma cell linestested at the times shown (i.e. day 1, 3 and 6) are reported in TableIV. The IC₅₀s of these two compounds, as well as those of 31 additionalcompounds (including those of Z-DTP2 derivatives such as mDTP3),calculated in KMS-11 and/or KMS-12 multiple myeloma cells at day 1, 3and 6 are reported in Table V.

As it can be seen in Table IV, Z-DTP1 and Z-DTP2 markedly decreased[³H]-TdR uptake in all the multiple myeloma cell lines tested in adose-dependent fashion (except that in the RPMI-8226 cell line, whichdisplay very low levels of Gadd45β; further discussed below; see FIG.12) (see also FIGS. 8A, 8B, 8C, and 12). Similar results were obtainedin these multiple myeloma cell lines when the IC₅₀s of Z-DTP1 and Z-DTP2were calculated using Trypan blue exclusion assays (measuring cellviability) (data not shown).

Results

As shown in Table IV, all the multiple myeloma cell lines testedexhibited high sensitivity to Z-DTP-afforded inhibition of cellsurvival/proliferation (see also FIGS. 8A, 8B, 8C, and 12). As in canalso be seen in Table IV, however, these cell lines displayed variablesensitivity to Z-DTP1 and Z-DTP2. Indeed, some cell lines were alreadyhighly sensitive to Z-DTP-afforded inhibition of cellsurvival/proliferation after a 24-hr treatment with these compounds(e.g. see the IC₅₀=1.3 μM of KMS-12 cells, and the IC₅₀=2.88 μM ofKMS-11 cells Z-DTP2 at 24 hrs), and all of them were highly sensitive toboth Z-DTP1 and Z-DTP2 after treatment for 144 hrs, with IC₅₀s rangingfrom 10.1 nM to 4.9 μM for Z-DTP1, and from 10 nM to 4.5 μM for Z-DTP2,at this time point (Table IV). Of note, the Z-DTP2 derivative, mDTP3(compound 17), was tested in KMS-11 and KMS-12 cell lines, and showed animproved cellular activity in these cell lines compared to Z-DTP1 andZ-DTP2, with IC₅₀s of 16 nM and 25 nM, respectively, at day 6, comparedto the IC₅₀s of Z-DTP1 (i.e. 316 nM and 10.1 nM, respectively) andZ-DTP2 (i.e. 66 nM and 10 nM, respectively) (see Table V) (see alsoFIGS. 20A, 20B, and 20C). Hence, the active DTPs, but not negativecontrol Z-DNCs, have strong cytotoxic activity in most multiple myelomacell lines. Furthermore, our most recent derivatives (e.g. mDTP3) retainhigh potency in vitro, but show improved cellular activity in multiplemyeloma cells, with substantially reduced MW (˜500 versus >700), henceincreased ligand efficiencies (see Table V) (see also FIG. 13).

Example 7 Restoration of Gadd45β-Inhibited MKK7 Catalytic Activity byTetrapeptides

Materials and Methods

In FIG. 6, transient transfection of pcDNA-FLAG-MKK7 in HEK-293 cellswas performed using the method of Ca₃(PO₄)₂ precipitation, essentiallyas described in Papa, S et al., (2004) Nat. Cell Biol. 6, 146-153 andreferences therein. 36 hrs after transfection, the cells were treatedwith 100 ng/ml phorbol 12-myristate 13-acetate (PMA) and 1 μM ionomycinfor 30 min at 37° C. Cell extracts were prepared as described in example4 and used for immunoprecipitation with anti-FLAG antibodies (binding toFLAG-MKK7) as described in Papa, S et al., (2004) Nat. Cell Biol. 6,146-153 and references therein. Briefly, 50 μg of cell lysate fromPMA-ionomycin (P11)-treated or untreated, HEK-293 cells transientlyexpressing FLAG-MKK7 was incubated with 10 μl of anti-FLAG M2 AffinityGel (SIGMA) for 4 hrs at 4° C. during rotation. The immunoprecipitateswere then washed 3 times in lysis buffer and twice more in kinase buffer(10 mM HEPES, 5 mM MgCl₂, 1 mM MnCl₂, 12.5 mM β-glycerophosphate, 2 mMDTT, 4 mM NaF and 0.1 mM Na₃VO₄). MKK7 catalytic activity was finallymeasured in kinase assays by incubating FLAG-MKK7 immunoprecipitates at30° C. for 20 min with 20 μl of kinase buffer containing 2 μM ofrecombinant GST-JNK1 and 5 μCi of [γ-³²P]ATP) (kinase reaction), asdescribed in Papa, S et a., (2004) Nat. Cell Biol. 6, 146-153 andreferences therein.

In some reactions, to test the ability of D-tetrapeptide antagonists todisrupt the Gadd45β-MKK7 interaction and so release the catalyticactivity of MKK7 from Gadd45β-afforded inhibition, FLAG-MKK7immunoprecipitates were 1) first pre-incubated for 10 min at 30° C. witheither 1 nM or 5 nM of DTP1, DTP2 or negative control (NC)D-tetrapeptides, NC1, NC2, NC3 and NC4, and 2) then incubated foranother 10 min at 30° C. with or without 5 μM of a GST-fusion protein ofrecombinant human (h)Gadd45β (GST-hGadd45β; purified from bacteriallysates as described in Papa, S., (2007) J. Biol. Chem. 282,19029-19041), before using them for the kinase reaction described above,as indicated in FIG. 6.

In all cases, kinase reactions were terminated by the addition ofLaemmli sample buffer. Proteins were then resolved by 10% SDS-PAGE, andMKK7 kinase activity revealed by autoradiography. For a furtherdiscussion of MKK7 kinase assay conditions, the reader is directed toPapa, S et al., (2007) J. Biol. Chem. 282, 19029-19041 and Papa, S etal., (2004) Nat. Cell Biol. 6, 146-153 and the references therein.

Results

Results are shown in FIG. 6 wherein the intensity of a band correspondsto the degree of MKK kinase activity measured, as this intensity isproportional to the amount of [γ-³²P]ATP) incorporated by MKK7 into itssubstrate, GST-JNK1. As can be seen from FIGS. 3C, 4, and 5, incubationwith DTP1 or DTP2 effectively and specifically disrupted the Gadd45βinteraction with MKK7 and as a consequence, as can be seen from FIG. 6,fully restored the catalytic activity of MKK7, whereas incubation withnegative control (NC) tetrapeptides NC1, NC2, NC3 or NC4 did not (FIG.6, top panels). It can also be seen from FIG. 6 that neither the controltetrapeptides, NC1, NC2, NC3 and NC4, nor the active tetrapeptides, DTP1and DTP2, afforded any inhibition of MKK7 catalytic activity whenincubated with MKK7 in the absence of recombinant GST-hGadd4513 (FIG. 6,bottom panels). These results are consistent with those shown in FIGS.3C, 4, and 5, where only DTP 1 and DTP2, but not NC1, NC2, NC3 or NC4were capable of disrupting the MKK7-Gadd45β interaction in either ELISAor co-immunoprecipitation assays.

Example 8 Specific Killing of Tumour Cell Lines Featuring ConstitutiveNF-κB Activity and/or High Levels of Gadd45β Expression by Tetrapeptides

Materials and Methods

This example investigates the use of control tetrapeptides (that isZ-DNC, Z-LNC, and Ac-DNC) and in vitro bioactive lead tetrapeptides(that is Z-DTP1, Z-DTP2, Z-LTP2 and Ac-DTP2) for the killing of a largepanel of human and murine tumour cell lines of various tissues oforigin. The tumour cell lines tested include: the multiple myeloma celllines U266, KMS-11, NC1-H929, ARH-77, JJN-3, KMS-12, KMS-18, KMS-27,RPMI-8226; the diffuse large B-cell lymphoma cell lines LY-3 and SUDHL6;the Burkitt's lymphoma cell lines BJAB, ST486, RAJI, RAMOS, Namalwa, andHS-SULTAN; the pro-monocytic leukaemia cell line U937; the T-cellleukaemia and lymphoma cell lines JURKAT, HUT-78, MT-2, MT-4, MOLT4,MT2-HTLV-I, and CEM; the breast cancer cell lines MCF7, MD-MDA-231, andMD-MDA-486; the pre-B-cell lymphoma cell lines NALM-6 (human) and 70Z/3(mouse); the chronic myelogenic leukemia cell line K652; the B-celllymphoma cell lines KARPAS (human) and A20 (mouse); the human embryonickidney cell line HEK-293T. Tumour cell lines were cultured as describedpreviously (Zazzeroni et al. 2003, Blood 102: 3270-3279) in RPMI-1640(multiple myeloma, diffuse large B-cell lymphoma, Burkitt's lymphoma,pro-monocytic leukaemia, T-cell leukaemia and lymphoma, pre-B-celllymphoma, chronic myelogenic leukemia, and B-cell lymphoma cell lines)or DMEM medium (breast cancer and embryonic kidney cell lines)supplemented with 10% fetal bovine serum (FBS), 1% glutamine, 100 U/mLpenicillin, and 100 μg/mL streptomycin in a humidified atmosphere of 5%CO₂ at 37° C.

For proliferation inhibition assays (FIGS. 7, 8 and 9) and cell deathassays (FIGS. 10A, 10B, 10C, 10D and 10 E), cells were seeded in wellsof 96-well plates at a concentration of 1.0×10⁴ cells/ml (proliferationassays) or in wells of 24-well plates at a concentration of 4×10⁵cells/ml (apoptosis assays) and cultured for up to 6 days. During thistime, cells were cultured in medium alone (untreated cultures) or inmedium supplemented with either control (e.g. Z-DNC) or active (e.g.Z-DTP2) tetrapeptides (treated cultures) to achieve a finalconcentration of the tetrapeptides in the cultures of 10 μM or 100 μM,as indicated. For the proliferation inhibition assays aimed at assessingthe effects of the tetrapeptides on survival/proliferation of tumourcells, cultures were analyzed daily by either Trypan blue exclusion(discriminating between live and dead cells) and cell counting (data notshown) or [³H]thymidine incorporation assays (FIGS. 7A, 7B, 7C, 8A, 8B,8C and 9), as indicated. In these latter assays, the effects of Z-DTPs,Ac-DTPs and Z-LTPs on the survival/proliferation of tumour cell lineswas investigated by measuring DNA synthesis using standard tritiatedthymidine ([³H]thymidine; [³H]-TdR) uptake assays. In the analysesshown, cells were incubated for 24, 72, 120 or 144 hrs at 37° C. in thepresence or absence of control or bioactive tetrapeptides, as indicated,then subjected to an additional incubation for 18 hrs with [³H]-TdR(0.037 MBq/well, equivalent to 0.5 μCi/well). Cells were subsequentlyharvested onto glass fibre filter mats using a 96-well plate automatedcell harvester, after which scintillation fluid was added, and[³H]thymidine incorporation measured by liquid scintillationspectroscopy on a beta counter. The results are expressed as thepercentages of the counts per minute (c.p.m.) (directly correlating withthe extent of cell proliferation) measured with tetrapeptide-treatedcultures relative to the c.p.m. measured with the corresponding culturesincubated with medium alone (untreated cells). All experiments wereperformed in triplicate. As it is further discussed later, Z-DTP2,Z-LTP2 and Ac-DTP2 yielded similar results to Z-DTP1, Z-LTP1 andAc-DTP1, respectively, in these survival/proliferation assays, albeitZ-DTP2 exhibited a slightly higher activity than Z-DTP1 (data not shown;see also Table IV).

Cell apoptosis in cultures was measured at the times indicated by theuse of propidium iodide (PI) nuclear staining and flow cytometry (FC)preformed essentially as described previously (Riccardi and Nicoletti(2006) Nature Protocols 1, 1458-1461) in order to identify cells with asub-G₁ DNA content (i.e. apoptotic cells) (FIGS. 10A, 10B, 10C, 10D and10E). For these assays, cells (4×10⁵ cells/ml) were cultured in 24-wellplates for 72 or 144 hrs as indicated in FIGS. 10A, 10B, 10C, 10D and10E, then washed twice in 1× phosphate buffer saline (PBS) and fixedwith 70% ice-cold ethanol for 16 hrs at −20° C., after which they weresubjected to centrifugation and subsequently resuspended in 1×PBScontaining 100 μg/mL of RNAase A. After this step, the cells wereincubated at room temperature for 30 min and subjected tocentrifugation, then resuspended in 50 μg/mL of PI, and incubated foranother 45 min at 4° C. in the dark. Flow cytometry (FC) was finallyperformed using a FACsCalibur automated system, and the data wereanalyzed using the FlowJo software.

In order to determine the basis for the different sensitivity of tumourcell lines to Z-DTP-induced killing, we measured levels of Gadd45βexpression in a panel of 29 tumour cell lines or different tissues oforigin by using quantitative real-time polymerase chain reaction(qRT-PCT) and correlated these levels with the degree of susceptibilityof these cell lines to the cytotoxic activity of Z-DTPs. For theseanalyses, which are shown in FIG. 12, the breast cancer and HEK-293Tcell lines were cultured in 75 cm² flasks (5×10⁶ cells/flask) incomplete DMEM medium, whereas all the other cell lines were cultured inwells of 6-well plates at 5×10⁵ cells/well in complete RPMI-1640 mediumas described above. Total RNA was extracted with Trizol and purifiedusing the PureLike RNA mini-kit (Invitrogen). 1 μg of RNA was added astemplate to reverse-transcriptase (RT) reactions performed using theGeneAmp RNA PCR Kit (Applied Biosystems). qRT-PCRs were carried out withthe resulting cDNAs in triplicate using SYBR Green PCR Master Mix(Applied Biosystems), the Gadd45β-specific primers listed in Table VIIand an ABI 7900 real-time PCR machine. Experimental Ct values werenormalized to β-actin, and relative mRNA expression calculated versus areference sample (i.e. mRNA from HEK-293T cells). The sensitivity ofcancer cell lines to Z-DTP-induced killing was analyzed as describedabove by performing [³H]thymidine incorporation assays after treatmentof the cells with 10 μM of Z-DTP2 for 144 hrs. Also shown in FIG. 12 isthe correlation plot of mRNA Gadd45β expression versus the percentage ofcell survival after treatment with Z-DTP2. The significance of thecorrelation coefficient between the 2 parameters' domain was calculatedby Pearson correlation, which quantifies the association between twovariables, using the GraphPad software.

In order to determine whether Z-DTP-induced killing of cancer cell lineswas due to the induction of cytotoxic JNK signalling, we monitored JNKactivation after treatment of two representative, sensitive multiplemyeloma cell lines (i.e. the KMS11 and NCI-H929 cell lines) with Z-DTP2(FIG. 11). To this end, we used Western blots analyses for an assessmentof JNK phosphorylation—an indicator of JNK activation. The KMS11 andNCI-H929 multiple myeloma cell lines were cultured in 6-well plates at5×10⁵ cells/well in complete RPMI-1640 medium as described above, andtreated with 10 μM of Z-DTP2 or of the negative control tetrapeptide,Z-DNC, for 3, 6, 12 or 24 hrs (FIG. 11). After tetrapeptide treatment,cell lysates were prepared essentially as described in Example 4 andWestern blots were performed using an anti-phospho(P)-JNK-specificantibody. The methodology used for Western blot analyses is described inthe references by De Smaele, et al. (2001) Nature 414:306-313; Papa, Set al., (2004) Nat. Cell Biol. 6, 146-153; Papa, et al. 2007 J. Biol.Chem. 282:19029-19041; Papa, et al. (2008) J. Clin. Invest.118:191-1923. β-actin levels were determined using a β-actin-specificantibody and served as loading control (FIG. 11). TNFα stimulation(2,000 U/ml) of KMS11 and NCI-H929 cells was carried out for 5, 10 or 30min and used as positive control for JNK activation (FIG. 11). Theseanalyses revealed that JNK activation is only caused by treatment withZ-DTP2, but not by treatment with Z-DNC. Similar effects of Z-DTP2 wereseen on MKK7 activation (data not shown). Importantly, as seen with thebiological activity of Gadd45β (see references: De Smaele, et al. (2001)Nature 414:306-313; Papa, S et al., (2004) Nat. Cell Biol. 6, 146-153;Papa, et al. 2007 J. Biol. Chem. 282:19029-19041; Papa, et al. (2008) J.Clin. Invest. 118:191-1923), the effects of Z-DTP2 in multiple myelomacells were specific for the MKK7/JNK pathway, as this compoundsexhibited no effect on the activation of the IKK/NF-κB, ERK and p38pathways (data not shown).

Results

FIGS. 7A, 7B and 7C show that Z-protected derivatives of DTP2 (Z-DTP2),but not of acetly derivatives (Ac-DTP2) or of L-isomers of Z-DTP2(Z-LTP2), nor the negative control tetrapeptides, Z-DNC, Ac-DNC andZ-LNC, markedly inhibit the proliferation of three representativemultiple myeloma cell lines out of the 8 susceptible multiple myelomacell lines tested (i.e. U266, KMS-11, and NCI-H929), of the Burkitt'slymphoma cell line, BJAB, and of the pro-monocytic leukemia cell line,U937 (see also FIGS. 8 and 12, and Table IV; additional multiple myelomalines). The cells were cultured with 10 μM of either Z-DTP2 or Z-DNC(FIG. 7A), Ac-DTP2 or Ac-DNC (FIG. 7B), and Z-LTP2 or Z-LNC (FIG. 7C) asindicated. [³H]-tTdR incorporation (measuring DNA synthesis) of treatedcells was measured and compared to that of cells cultured with mediaalone. The data are expressed as the percentage of c.p.m. observed withtumour cells after treatment with Z-DTP2, Ac-DTP2 or Z-LTP2 (filledcolumns), or with Z-DNC, Ac-DNC or Z-LNC (empty columns) relative to thec.p.m. measured with cells cultured with medium alone (untreated cells).A marked inhibition of cell proliferation was observed in multiplemyeloma and other tumour cell lines treated with Z-DTP2, but not inthose treated with Z-DNC. In FIG. 7B, the absence of Ac-DTP2'stumoricidal activity in multiple myeloma cell lines correlated with thelow cellular permeability of this compound, as established in CaCO2assays (data not shown). The viability of multiple myeloma cell linesafter treatment with other, less effective DTP derivatives (alsodesigned to improve DTPs' cellular uptake), including those bearing amethyl (Me), acetyl (Ac), myristyl (Myr), 3-methoxy,4-hydroxy-benzoyl,benzoyl, 6Cl-benzyloxycarbonyl (6Cl-Z), and/orfluorenylmethyloxycarbonyl (Fmoc) group, is not shown. Although Z-LTPs'in vitro potency and cellular uptake were comparable to those of Z-DTPs(see FIG. 3C; also data not shown), Z-LTP2 showed low activity inmultiple myeloma cells (FIG. 7C), due to low stability in biologicalfluids (see FIG. 4). A similar inhibition of cell proliferation wasobserved in the tumour cell lines treated with Z-DTP1, but not in thosetreated with Ac-DTP1 or Z-LTP1 (data not shown)—despite that (as alsoseen with DTP2 derivatives) these two latter compunds exhibitedcomparable potency to Z-DTP1 in vitro (see FIGS. 3C and 4). Together,these data establish the high cytotoxic activity of Z-DTPs (FIG. 7A anddata not shown) compared to the inactivity of Ac-DTPs (FIG. 7B and datanot shown) and the low activity of Z-LTPs (FIG. 7C and data not shown).

In FIGS. 8A, 8B and 8C, we examined the effects of D-tetrapetidetreatment on the proliferation of a larger panel of multiple myelomacell lines (i.e. U266, KMS-11, JJN-3, NCI-H929, ARH-77, KMS-27, KMS-18,KMS-12, and RPMI-8226). Other tumour cell lines tested include thediffuse large B-cell lymphoma cell lines, LY-3 and SUDHL6, the Burkitt'slymphoma cell lines, BJAB, ST486 and RAJI, and the pro-monocyticleukemia cell line, U937. The cells were treated with 10 μM of eitherZ-DTP2, Z-DTP1 or Z-DNC, as shown, for the times indicated (i.e. 24, 72or 144 hrs). [³H]Thymidine incorporation of treated cells was measuredas in FIG. 7 and compared to that of cells cultured with media alone.The data are expressed as the percentage of c.p.m. observed with tumourcells treated with Z-DTP2 or Z-DTP1 (filled columns), or with Z-DNC(empty columns) relative to the c.p.m. measured with untreated cells.FIG. 8A shows that Z-DTP2, but not Z-DNC, markedly inhibits thesurvival/proliferation of 8 out of 9 multiple myeloma cell lines tested(i.e. U266, KMS-11, JJN-3, and NCI-H929, ARH-77, KMS-27, KMS-18,KMS-12), of the Burkitt's lymphoma cell line, BJAB, of the diffuse largeB-cell lymphoma (DLBCL) cell line, LY-3, and of the pro-monocyticleukemia cell line, U937 (see also FIG. 12). Notably, Z-DTP2 showedcytotoxic activity only in the DLBCL cell line of the activated-B-cell(ABC)-like subtype (i.e. LY3), which depends on NF-κB for survival, andnot in that of the germinal center B-cell (GCB)-like (i.e. SUDHL6)subtype, which does not feature constitutive NF-κB activation (Ngo V N,et al. Nature 441(7089):106-10; see also FIG. 12). It also showed potentcytotoxic activity of Z-DTPs in the vast majority of the multiple celllines tested—all of which depend on NF-κB for survival. As shown inFIGS. 8B and 8C, the inhibitory effects of Z-DPT1 and Z-DTP2 on tumourcell proliferation increased with time—maximal inhibition ofproliferation was observed after tetrapeptide treatment for 144 hrs,albeit these effects were already apparent after treatment for 24 hrs.These data are in agreement with those obtained by cell counting intrypan blue exclusion assays (data not shown) and in PI nuclear stainingassays for DNA content (see FIGS. 10A, 10B, 10C, 10D and 10E); also datanot shown). Together, these and other data show that Z-DTPs' cytotoxicactivity is selective for tumour cells exhibiting constitutive NF-κBactivity (see also FIG. 12; also data not shown).

The specificity of the cytotoxic activity of Z-DTPs was furthercorroborated by the [³H]thymidine proliferation assays shown in FIG. 9.This Figure shows the absence of Z-DTP2-induced cytotoxicity in a panelof 22 resistant tumour cell lines after treatment for 144 hours, evenwhen this compound was used at very high concentrations—that is 100 μM.The [³H]thymidine proliferation assays shown in FIG. 9 were performed asdescribed in FIG. 8.

As it can be seen in FIG. 9, Z-DTP2 exhibited no cytotoxicity in theT-cell leukaemia and lymphoma cell lines, JURKAT, HUT-78, MT-2, MT-4,MOLT4, MT2-HTLV-I, and CEM, the Burkitt's lymphoma cell lines BJAB,ST486, RAJI, RAMOS, Namalwa, and HS-SULTAN, the breast cancer cell linesMCF7, MD-MDA-231, and MD-MDA-486, the pre-B-cell lymphoma cell linesNALM-6 and 70Z/3, the B-cell lymphoma cell lines KARPAS and A20, thechronic myelogenic leukemia cell line K652, the human embryonic kidneycell line HEK-293T, and the multiple myeloma cell line RPMI-8226 (seealso FIG. 12). Also shown in FIG. 9 are the sensitive cell lines BJAB(Burkitt's lymphoma), KMS-11 and KMS-12 (multiple myeloma). Notably,there was a strong correlation in these cell lines also betweensensitivity to Z-DTP2-induced killing and levels of endogenous Gadd45βexpression (see FIG. 12). Of note, the RPMI-8226 cell line—the onlymultiple myeloma cell line tested that is resistant to Z-/mDTP-inducedkilling (FIGS. 8A and 9)—displayed very low levels of Gadd45β (see FIG.12), further confirming that the cytotoxic activity of DTPs in cancer isdependent on the levels of constitutive Gadd45β expression.

In FIGS. 10A, 10B, 10C, 10D and 10 E, the embedded panels show thepercentage of cells exhibiting propidium iodide (PI) staining indicativeof a sub-G₁ amount of DNA (i.e. cells which are either dead or dying byapoptosis), after treatment for the indicated times (i.e. 72 or 144 hrs)with either culture medium alone (untreated) or culture mediumdelivering a 10 μM final concentration of either Z-DTP2 or Z-NC1. Thepercentages of apoptotic cells are depicted in the histograms. Shown arethe five representative sensitive multiple myeloma cell lines, NCI-H929,(see FIG. 10A) KMS-11, (see FIG. 10B), ARH-77, (see FIG. 10C), JJN-3,(see FIG. 10D) and U266 (see FIG. 10E). As it can be seen,Z-DTP2-induced killing of multiple myeloma cells is due to thetriggering of apoptosis, and the portion of apoptotic cells seen aftercell exposure to this compound increases with the time of treatment.

FIG. 11 shows that Z-DTP2 treatment causes strong activation of JNK inmultiple myeloma cell lines. The two representative sensitive multiplemyeloma cell lines, KMS11 and NCI-H929, were treated with 10 μM ofZ-DTP2 or Z-DNC, as shown. JNK activation was monitored at the indicatedtimes by western blotting using an anti-phospho(P)-JNK-specificantibody. It can be seen that JNK phosphorylation (a marker of JNKactivation) only increases after treatment with Z-DTP2, but not aftertreatment with Z-protected negative control peptide (Z-DNC). Indeed,Z-DTP2 caused an even stronger activation of JNK than TNFα stimulationdid (2,000 U/ml)—our positive control. Similar effects of Z-DTP2 wereseen on MKK7 activation and using kinase assays to monitor JNK and MKK7activities (data not shown). Notably, as seen with the biologicalactivity of Gadd45β (see references: De Smaele, et al. (2001) Nature414:306-313; Papa, S et al. (2004) Nat. Cell Biol. 6, 146-153; Papa, etal. (2007) J. Biol. Chem. 282:19029-19041; Papa, et al. (2008) J. Clin.Invest. 118:191-1923), the effects of Z-DTP2, as well as of Z-DTP1 andmDTP3 (data not shown), in multiple myeloma cells were specific for theMKK7/JNK pathway, as no effects were observed with these compounds onthe IKK/NF-κB, ERK and p38 pathways (data not shown). Importantly,Z-DTPs' treatment failed to activate JNK in the multiple myeloma cellline, RPMI-8226, which is resistant to Z-DTP-induced killing (see FIGS.8A and 9). These and other data (see also FIG. 20) support the view thatZ-DTPs inducing apoptosis in tumour cell lines by activating JNKcytotoxic signaling.

Crucially, the data presented in FIGS. 12A and 12B show that thesensitivity of cancer cell lines to Z-DTP-induced killing correlateswith a very high degree of statistical significance with levels ofendogenus Gadd45β expression (p<0.01). Gadd45β mRNA expression wasassessed in a panel of 29 cancer cell lines by using qRT-PCR assays(FIG. 12A, top panel, red columns). Values were normalized to β-actin.Viability/proliferation in the same cancer cell lines was determined byperforming [³H]thymidine incorporation after treatment with 10 μM ofZ-DTP2 for 144 hrs. These results are shown in the bottom panel of FIG.12A (black columns). The values reported here represent the percentageof c.p.m. measured with cells treated with Z-DTP2 relative to the c.p.m.measured with untreated cells. FIG. 12B shows the correlation plot ofGadd45β expression versus the percentage of cell survival/proliferationobserved after treatment with Z-DTP2 for the same experiment shown inFIG. 12A. As it can be seen, the significance of the correlationcoefficient between the 2 parameters' domain is very high (p<0.01)(Pearson correlation, which quantifies the association between twovariables, calculated using the GraphPad software). This is a key issuefor the development of a successful therapy in man. These datademonstrate the high target specificity of the Z-DTPs in cells forGadd45β. In further support of this conclusion, sh-RNA-mediatedsilencing of Gadd45β induces apoptosis in multiple myeloma cells,whereas sh-RNA-mediated silencing of MKK7 MKK7 renders these cellscompletely resistant to Z-DTP-induced killing (see FIGS. 16, 17, 18, and20). Together, these data also show that should DTP-based therapy enterthe clinic, it will be possible to predict patient responder populationsvia simple and cost-effective qRT-PCR analysis. Accordingly, it followsthat primary cell from multiple myeloma patients can be analyzed forlevels of Gadd45β expression, and patients with high levels of thisexpression can be deemed as those who will receive the most benefit fromtreatment with the compounds of the invention. Hence, an importantaspect of the invention is a theranostic aspect—that is the applicationof a clinically useful assay to predict DTPs' therapy response inpatients.

Example 9 IC₅₀s In Vitro and in Cells of a Panel of Z-DTPs' Derivatives

We have developed an extensive plan of lead optimization to deliver asafe and effective new therapy for treating cancer and other diseasesand disorder, using our current leads as starting points. Z-DTP2 alreadyshows high stability, high solubility, sub-nM activity in vitro, andgood activity in multiple myeloma cells (primary and cell lines) andother cancer cells, with high target specificity and no toxicity innormal cells (see FIGS. 3C, 4, 8, 9, 12, 14 and 15; see also Table IV;data not shown). It also exhibits excellent starting DMPK and safetyprofiles in vivo (single i.v. bolus dose in mice) (see Tables VIII andIX). We have applied rational molecular design to produce DTPderivatives with improved ADMET properties whilst retaining highbioactivity (Geeson M P. 2008 J Med Chem. 51:817-834). In this approach,we have modified size (MW), lipophilicity (Log P), and ionization state(molecular charge)—the key bulky properties of molecules that influenceADMET properties—using our model pharmacophore to preserve structuralelements responsible for bioactivity in vitro (see FIG. 13). Asdescribed in this Example, our most recent derivatives (e.g. mDTP3 andmDTP4) retain high potency in vitro, but show improved killing activityin multiple myeloma cells, with substantially reduced MW (˜500 vs>700),hence increased ligand efficiencies (FIG. 13). We have also appliedadditional means of improving peptides' cellular activity and PK values,including cyclization, addition of blocking groups to internalizevulnerable amides, and/or replacement with non-amide linkages.

Materials and Methods

33 compounds were designed on the basis of the lead tetrapeptidesequences: Tyr-Glu-Arg-Phe and Tyr-Asp-His-Phe derived from the libraryscreening (see FIG. 3). All compounds—except for compounds 2, 3, 4, 5,6, 7, 10, 11, 12, 13, 14, 15, and 16 (see Table V)—were prepared by asolid phase method following classical Fmoc/tBu chemistry (as describedin the reference by Fields G B, Noble R L. Solid phase peptide synthesisutilizing 9-fluorenylmethoxy-carbonil amino acids. Int J Pept ProteinRes 1990; 35:161-214). Only amino acids in the D-configuration were usedto assemble, the peptides shown in Table V. N-terminal acetylation wascarried out by treatment with 10% acetic anhydride in dimethylformammide(DMF) containing 5% DIEA (di-isopropyl-ethylamine). Where necessary, theZ group was introduced by on-resin treatment with Z-OSu(Benzyloxycarbonyl-N-hydroxysuccinimide) 0.5 M in DMF/5% DIEA. Compoundswere cleaved from the resin using TFA (trifluoroacetic acid) andscavengers treatment, then were purified to homogeneity by preparativereverse phase (RP)-PLC. The synthesis of compounds 2, 3, 4, 5, 6, 7, 10,11, 12, 13, 14, 15, and 16 (Table V) was outsourced. Compound identityand purity was assessed by using LC-MS and NMR analyses. X₁ was benzoicacid, X₂ was benzylic acid, Y₁ was aniline, Y₂ was benzylamine, and Y₃was phenetylamine. The compounds were all dissolved in DMSO at the stockconcentration of 5 mM, and aliquots were then serially diluted in bufferto achieve the concentrations indicated for the ELISA competitionassays. Proteins were prepared as reported in Tornatore L., et al.(2008). J Mol Biol; 378:97-111.

The ELISA competition binding assays were performed as reported in thereference by Tornatore L., et al. (2008). J Mol Biol; 378:97-111 (seealso the Methods described in Examples 2 and 3), using peptides atincreasing concentrations, ranging between 0.01 nM and 10 nM. Briefly,GST-MKK7 was immobilized at 42 nM onto wells of 96-well microtiterplates. Competing compounds were preincubated with biotin-hGadd45β (21nM) and then incubated with the coated kinase. For each compound, theIC₅₀ in vitro was calculated as the concentration resulting in a 50%reduction of Gadd45β binding to MKK7 relative to the binding observed inthe absence of competitors.

We investigated the effects of each compound on theviability/proliferation of the DTP derivatives in the tworepresentative, sensitive multiple myeloma cell lines, KMS12 and KMS-11.[³H]Thymidine incorporation assays in KMS11 and KMS12 multiple myelomacells lines were performed as described for Examples 6 and 8 (FIGS. 7A,7B, 7C, 8A, 8B, and 8C, and Table IV. Briefly, cells in 96-wells platewere cultured and treated separately with the indicated compound inwells of in 96-wells plates using increasing compound concentrations,ranging between 0.1 nM and 10 μM. Cell cultures and compound treatmentswere also carried out as described for FIGS. 7A, 7B, 7C, 8A, 8B, and 8C,and Table IV. [³H]Thymidine upake, measuring cellviability/proliferation, was determined after treatment with thecompounds for 1, 3, o 6 days as indicated. At these times, the IC₅₀s ofeach compound were calculated as described in Example 6 by determiningthe concentration resulting in a 50% inhibition of cellsurvival/proliferation relative to the survival/proliferation observedwith untreated cells.

The IC₅₀s in vitro (ELISA) and in cells (KMS-11 and KMS-12 cells) of the33 compounds described in this Example are reported in Table V.

Results

Shown in Table V are the IC₅₀ values in vitro and in cells of a panel oftetra- and tripeptides designed on the basis of the consensus sequences,Tyr-Glu-Arg-Phe and Tyr-Asp-His-Phe, derived from the library screeningand lead optimization chemistry.

These compounds were screened in vitro using an ELISA competition assaywhere the displacement of the binding of biotin-Gadd45β to coatedGST-MKK7 was determined by testing the activities of the compounds atdifferent concentrations. In vivo IC₅₀s for a group of selectedcompounds were determined using a [³H]thymidine incorporation assays inKMS-11 and KMS-12 myeloma cells lines to assess the tumouricidalactivities of the compounds. IC₅₀s of the indicated compounds in cellswere determined after a treatment for 1, 3 or 6 days. Z denotes abenzyloxycarbonyl group. As it can be seen in Table V, the most activecompounds in cells were compound 9, denoted as Z-DTP2 (IC₅₀=10 nM inKMS-11 cells; IC₅₀=66 nM in KMS-12) and compound 17, denoted as mDTP3(IC₅₀=25 nM in KMS-11 cells; IC₅₀=16 nM in KMS-12).

The 33 compounds described in this Example were all screened in vitro,in ELISA competition assays, for their ability to disrupt theGadd45β/MKK7 interaction (Table V). Most of these compounds-except forcompounds 18, 20, 21, 22, 32, and 33-were also screened in cells, usinga [³H]thymidine incorporation assays in KMS-11 and/or KMS-12 multiplemyeloma cells lines, and their IC₅₀s in these cells determined at day 1,3 and 6. As it can be seen in Table V, compounds 1, 2, 3, 4, 5, 6, 7, 9,15 and 17 were tested in both cell lines. Compounds 15 and 19 were onlytested in KMS-12 cells. Compounds 10, 11, 12, 13, 14, 23, 24, 25, 26,27, 28, 29, 30, and 31 were only tested in the KMS-11 cell line.Compounds 18, 20, 21, 22, 32, and 33 were not tested in cells due totheir relatively low activity in vitro.

Table V shows that the IC₅₀s in vitro of the compounds tested rangedbetween 100 μM (see compound 7, X₂-Asp-His-Y₃; compound 15,X₂-Glu-Arg-Y₃; and compound 19, Z-Tyr-Arg-Phe) and >10 nM (see compounds24, 27, 30, 31, 32, and 33). As it can be seen, the activities of thecompounds that were in vitro were often reflected on their activities incells, although some of the compounds active in vitro had relatively lowactivity in cells, plausibly due to their poor cellular uptake, e.g.compare compound 15 (showing an in vitro IC₅₀=100 μM, and an IC₅₀=263 nMin KMS-11 cells) to compound 9 (Z-DTP2; showing an in vitro IC₅₀=190 μMand an IC₅₀=10 nM in KMS-11 cells). The data in cells also show that thepresence of a Z group at the N-terminus and/or of basic side chainsresulted in a higher activity in cells, due to increased cellularuptake. For examples of the relevance of the basic side chain, comparethe IC₅₀s in cells of compound 19 (Z-Tyr-Arg-Phe-NH₂; IC₅₀=81 nM inKMS-12 cells at day 3) to that of compound 8 (Z-Tyr-Asp-Phe-NH₂; IC₅₀>10μM in KMS-11 cells at day 3) (i.e. Arg to Asp exchange), or to that ofcompound 16 (Z-Tyr-Glu-Phe-NH₂; IC₅₀=3.0 nM in KMS-11 cells at day 3)(i.e. Arg to Glu exchange); also note the comparable, low IC₅₀s in vitroof these three compounds—all of which are in the sub-nM range (Table V).For examples of the relevance of the Z group, compare the IC₅₀s in U266,KMS-11, and NCI-H929 cells of Z-DTP2 (FIG. 7A) to that of Ac-DTP2 (FIG.7B); see also the similar IC₅₀s in vitro of these two compounds (FIGS.3C and 4; data not shown). The data also show that compounds withoutaromatic rings at both ends (e.g. compounds 20 and 21) are inactive bothin vitro and in cells (Table V), indicating that such aromatic rings areabsolutely required for bioactivity. Interestingly, the presence of 2tyrosines at the N-terminus also resulted in loss of activity (Table V).

Example 10 IC₅₀s In Vitro of a Panel of Additional Z-DTPs' Derivatives

Material and Methods

A panel of 18 additional compounds was designed on the basis of the leadtripeptide sequence, Tyr-Arg-Phe (i.e. mDTP3), in order to investigatethe relevance to bioactivity of: 1) the distance between the twoaromatic rings; 2) the properties of the amino acid in the centralposition; 3) the occurrence the acetyl group at the N-terminus; 4) andthe presence of substituents of the aromatic rings (see Table VI). Allcompounds were prepared by a solid phase method following classicalFmoc/tBu chemistry (as described in the reference by Fields G B, Noble RL. Solid phase peptide synthesis utilizing 9-fluorenylmethoxy-carbonilamino acids. Int. J. Pept. Protein Res. 1990; 35:161-214). N-terminalacetylation was carried out by treatment with 10% acetic anhydride indimethylformammide (DMF) containing 5% DIEA (di-isopropyl-ethylamine).

Compounds were cleaved from the resin by using TFA (trifluoroaceticacid) and scavengers treatment, then were purified to homogeneity bypreparative reverse phase (RP)-PLC. Compound identity and purity wereassessed by LC-MS and NMR analyses. Compounds were purified usingRP-HPLC, then all were dissolved in DMSO at the stock concentration of 5mM and stored until they were used. Aliquots were then serially dilutedin buffer to achieve the concentrations indicated in the ELISAcompetition assays. ELISA competition binding assays were performed asreported in the reference by Tornatore L., et al. (2008). J Mol Biol;378:97-111 (see also the Methods in Examples 2 and 3), using peptides atincreasing concentrations, ranging between 0.01 nM and 10 nM. Briefly,GST-MKK7 was immobilized at 42 nM onto wells of 96-well microtiterplates. Competing compounds were preincubated with biotin-hGadd45β (21nM) and then incubated with the coated kinase. For each compound, theIC₅₀ in vitro was calculated as the concentration resulting in a 50%reduction of Gadd45β binding to MKK7 relative to the binding observed inthe absence of competitors.

Results

Table VI shows the IC₅₀ values of a panel of 18 tripeptides anddipeptides designed on the basis of mDTP3 (Ac-D-Tyr-D-Arg-D-Phe).Compounds were designed to investigate the influence on bioactivity ofthe following parameters: 1) the distance between the two aromatic ringsat the N- and C-termini (see compounds A1, A1 bis, A3, A6, A7, and A8);2) the properties of the amino acid in the central position (seecompounds B2, B13, B16, B16 bis, 05, and 05 bis); 3) the presence orabsence of a hydroxyl group on the aromatic ring of the residues atpositions 1 and 3 (see compounds A9, O1, O3, O5, O5 bis, O6, O7, andO8); the occurrence of an acetyl group at the N-terminus (see compoundsA9 and O7; B16 and B16 bis; O1 and O8; O3 and O6; O5 and O5 bis).

The 18 additional compounds were tested for activity in vitro usingELISA completions assays and increasing compound concentrations, rangingfrom 0.01 nM to 100 nM. As it can be seen in Table VI, all thedipeptides tested were inactive regardless of the occurrence of a Phe orTyr amino acid at either the N-terminus or the C-terminus (see compoundsA1, A1 bis, A7 and A8). The introduction of a spacers longer than analpha-amino acid in the central position of the tripeptides alsoresulted in loss of activity in vitro (see compounds A3 and A6, carryinga β-alanine and an ε-caproic acid in the middle position, respectively).This was not true for tetrapeptides where positions Y₂ and Y₃ wereoccupied by Asp/Glu or His/Arg—compare the IC₅₀s in vitro of compound 9(i.e. Z-DTP2) to those of compound 16 (i.e. mDTP2), and those ofcompound 1 (i.e. Z-DTP1) to those of compound 8 (i.e.Z-Tyr-Asp-Phe-NH₂). This is because Z-DPT2 and Z-DTP1, which contain anexta-amino acid between the two active aromatic groups, retained highpotency in vitro (see IC₅₀s in Table V). Remarkably, as shown in TableVI, the removal of the hydroxyl group on the N-terminal tyrosine alsoresulted in the complete loss of bioactivity in vitro (see compounds A9,O1, O5, O5 bis, O7, and O8) regardless of the presence of an acetylgroup. Significantly, this observation points to an importantcontribution of the hydroxyl group to the interaction of the activecompounds with the target proteins. Indeed, this group is likelyinvolved in the formation of a H-bond or a polar interaction. Incontrast, the occurrence of a hydroxyl group on the aromatic ring at theC-terminus did not affect activity of the compounds (see compounds A9,O1, O3, O5, O5 bis, O6, O7 and O8). Likewise, replacing arginine withanother basic amino acid, such as histidine or lysine, or with prolinedid not alter bioactivity in vitro (see compounds B2, B13, B16, B16 bis,O5, and O5 bis), suggesting a minor role for the side chain of thisresidue in the ability of the compounds to disrupt the Gadd45β/MKK7interaction.

Example 11 Lentiviral Infections Establishing the Essential Role ofGadd45β in Multiple Myeloma Cell Survival

Material and Methods

To determine the role of Gadd45β and MKK7 in the survival of multiplemyeloma cell lines, we investigated the effects of down-regulating theexpression of Gadd45β or MKK7 in these cells (see FIGS. 16A, 16B, 16C,17A, 17B, 18A, 18B, 18C, 19A, 19B, and 19C). To this end, we performedinfection with lentiviruses expressing Gadd45β- and MKK7-targetingsh-RNAs, which result in the silencing of the Gadd45β and MKK7 genes,respectively. The DNA sequences encoding the targeting small hairpin(sh)-RNAs are listed in Table VII. The targeting sh-RNA sequences (i.e.sh-Gadd45β-1, sh-Gadd45β-2, sh-Gadd45β-3, sh-MKK7-1, and sh-MKK7-2) andthe non-specific control sequences, sh-NS-1 and sh-NS-2, were introducedbetween the BamHI and HpaI restriction sites of the lentiviral vector,LentiLox3.7 (see the reference by Yang et all 2006 PNAS 103,10397-10402). The production of high-titer lentiviral preparation inHEK-293T cells were performed using essentially the same conditionsdescribed in the references by Pham et all 2004 Cell 116, 529-542 and byYang et all 2006 PNAS 103, 10397-10402. For introduction of the Gadd45β-and MKK7-targeting sh-RNA sequences and the non-specific control sh-RNAsequences, the five representative Z-DTP-sensitive multiple myeloma celllines, ARH-77, NCI-H929, U266, KMS11 and KMS12, and the Z-DTP-resistantmultiple myeloma cell line, RPMI-8226, were infected with LentiLox3.7lentiviruses, as reported in published protocols essentially asdescribed in the reference by Yang et all 2006 PNAS 103, 10397-10402. 5days after infection, eGFP multiple myeloma cells were sorted using a BDFACSAria™ II cell sorter, then left to rest for 2 days before beginningthe analyses of cell survival and cell proliferation. The viability ofthe infected multiple myeloma cells was monitored over a period of 8days by performing flow cytometry-measuring the expression of enhancedgreen fluorescent protein (eGFP) (labelling infected cells)—and cellcounting (FIGS. 16A, 16B, 16C, 17A, 17B). Apoptosis (FIGS. 18A, 18B, and18C) and cell cycle distribution (FIGS. 19A, 19B, and 19C) were measuredby performing PI nuclear staining assays as described in Riccardi C. andNicoletti I 2006 Nature Protocols 1, 1458-1461 (see also the Methodsdescribed in Example 8).

Results

FIGS. 16A, 16B, and 16C show that the sh-RNA-mediated silencing ofGadd45β expression results in the rapid incution of cell death, leadingto reduced proliferation, in the representative Z-DTP-sensitive multiplemyeloma cell lines ARH-77 (FIG. 16A) and NCI-H929 (FIG. 16B), but not inthe Z-DTP-resistant multiple myeloma cell line, RPMI-8226 (FIG. 16C). Inthe experiment shown in FIGS. 16A, 16B, and 16C, multiple myeloma celllines were infected with lentivirus expressing either Gadd45β -specificsh-RNAs (sh-Gadd45β-1, sh-Gadd45β-2, or sh-Gadd45β-3), MKK7-specificsh-RNAs (sh-MKK7-1 or sh-MKK7-2), or non-specific sh-RNAs (sh-NS-1 orsh-NS-2), and viability of infected cells was monitored over a period of8 days by using flow cytometry—revealing cells expressing enhanced greenfluorescent protein (eGFP), that is infected cells—and cell counting.Shown is the percent survival of eGFP⁺ (that is lentivirus-infected)multiple myeloma cells at the times indicated relative to the viabilityof eGFP⁺ multiple myeloma cells in the same culture at day 0. Cells wereinfected with pLentiLox.3.7 lentiviruses expressing the indicatedsh-RNAs and eGFP, using standard methods (as reported in Yang H et al.,Proc Natl Acad Sci USA. 2006 Jul. 5; 103(27):10397-402). 5 days later,eGFP⁺ cells were sorted using a BD FACSAria™ II cell sorter, then leftto rest for 2 days before beginning the analyses of cell viability. Thistime (that is the start of the viability analyses) is denoted in FIGS.16A, 16B, and 16C as day 0. The data show that the inhibition of Gadd45βexpression, but not the inhibition of MKK7 expression, rapidly causescell death in multiple myeloma cell lines that are sensitive toZ-DTP-induced toxicity (that is the ARH-77 and NCI-H929 cell lines)(FIGS. 16A and 16B, respectively), but not in the RPMI-8226 multiplemyeloma cell line (FIG. 16C), which is resistant to this toxicity. Thesedata further establish the target specificity of Z-DTPs for theGadd45β/MKK7 complex in multiple myeloma cells (see also FIGS. 7, 8, 9,and 12; killing and qRT-PCR assays). Indeed, in further agreement withthis conclusion, the kinetics of the inhibition of multiple myeloma cellproliferation observed after the silencing of Gadd45β were very similarto those observed after treatment of these cells with Z-DTPs (see FIGS.7A, 8B, and 8C). The data also demonstrate the essential role thatGadd45β plays in multiple myeloma cell survival, thus further validatingGadd45β as a therapeutic target in multiple myeloma.

FIGS. 17A and 17B showing that the sh-RNA-mediated silencing of Gadd45β,but not that of MKK7, has potent inhibitory activity on thesurvival/proliferation only of multiple myeloma cell lines that aresusceptible to Z-DTPs-induced killing (e.g. the ARH-77 and NCI-H929 celllines; see also FIGS. 7 and 8, sensitivity to Z-DTP-induced killing). Instriking contrast, the viability of the Z-DTP-resistant multiple myelomacell line, RPMI-8226, was completely unaffected by sh-RNA-mediatedGadd45β inhibition. Cell proliferation/survival in FIGS. 17A and 17Bwere determined by the use of [³H]Thymidine incorporation assays,performed as described in Examples 6 and 8. Shown in FIG. 17A is theviability of the three representative multiple myeloma cell lines,RPMI-8226, NCI-H929 and ARH-77, after the silencing of Gadd45β or MKK7.FIG. 17B shows the viability/proliferation of the multiple myeloma cellline, ARH-77, after the silencing of Gadd45β or MKK7 using threedifferent Gadd45β-specific sh-RNAs (sh-Gadd45β-1, sh-Gadd45β-2, orsh-Gadd45β-3), two different MKK7-specific sh-RNAs (sh-MKK7-1 orsh-MKK7-2), and two different non-specific sh-RNAs (sh-NS-1 or sh-NS-2).Multiple myeloma cell lines were infected with the indicatedsh-RNA-expressing pLentiLox.3.7 lentivirus, then eGFP⁺ multiple myelomacells (that is cells infected with lentivirus) were sorted using a BDFACSAria™ II cell sorter as in FIG. 16. The [³H]thymidine incorporationassays depicted in FIGS. 17A and 17B were performed 10 days after cellsorting, corresponding to day 8 in FIG. 16. Shown is the percent[³H]thymidine incorporation (that is c.p.m.), a measure of cellproliferation, at day 8 (that is 10 days after cell sorting) relative tothe [³H]thymidine incorporation occurring in the same cells at day 0(that is 2 days after cell sorting). These data further establish thetarget specificity of Z-DTPs for the Gadd45β/MKK7 complex in multiplemyeloma cells (see also FIGS. 7, 8, 9, 12, and 16), and confirm theessential role that Gadd45β plays in multiple myeloma cell survival.Together, they also further validate Gadd45β as therapeutic target inmultiple myeloma.

FIGS. 18A, 18B, and 18C show that the sh-RNA-mediated silencing ofGadd45β effectively induces apoptosis in the Z-DTP-sensitive multiplemyeloma cell lines, ARH-77 (FIG. 18A) and NCI-H929 (FIG. 18B), but notin the Z-DTP-resistant multiple myeloma cell line, RPMI-8226 (FIG. 18C)(see also FIGS. 16 and 17, sh-RNA-mediated silencing; FIGS. 7, 8, and12, multiple myeloma cell line sensitivity to Z-DTP-induced killing andlevels of Gadd45β expression). Apoptosis induction in FIGS. 18A, 18B,and 18C was determined by the use of PI nuclear staining assays,performed as described in Example 8. These data demonstrate that theinhibition of multiple myeloma cell survival/proliferation caused by thedown-regulation of Gadd45β expression observed in FIGS. 16 and 17 wasdue to the induction of programmed cell death mediated by the apoptosispathway. Notably, no significant induction of apoptosis was observed inthe same multiple myeloma cell lines in the absence of lentiviralinfection (uninfected) or after expression of MKK7-specific sh-RNAs(sh-MKK7-1 and sh-MKK7-2) or non-specific sh-RNAs (sh-NS-1 and sh-NS-2)(FIGS. 18A, 18B, and 18C). Multiple myeloma cell lines were infectedwith sh-RNA-expressing pLentiLox.3.7 lentiviruses, and eGFP⁺ multiplemyeloma cells (that is cells infected with lentivirus) were sorted usinga BD FACSAria™ II cell sorter as in FIG. 16. PI nuclear staining assayswere performed 10 days after cell sorting, corresponding to day 8 inFIG. 16. The percentages of apoptotic cells (that is cells exhibitingsub-G₁ DNA content) are depicted in the histograms. Importantly, thelevels of apoptosis induced by the different Gadd45β-specific sh-RNAs(that is sh-Gadd45β-1, sh-Gadd45β-2, and sh-Gadd45β-3) correlated withthe levels of Gadd45β downregulation induced by each of theseGadd45β-specific sh-RNAs (FIG. 18A; also data not shown). The data inFIGS. 18A, 18B, and 18C further establish the target specificity ofZ-DTPs for the Gadd45β/MKK7 complex in multiple myeloma cells (see alsoFIGS. 7, 8 and 9, killing assays with Z-DTPs; FIG. 12, statisticallysignificant correlation between Gadd45β expression and cancer cellsensitivity to Z-DTP-induced killing; FIGS. 16 and 17, induction ofmultiple myeloma cell line killing by the downregulation of Gadd45β, butnot of MKK7), and confirm the essential role that Gadd45β plays inmultiple myeloma cell survival. Together, they further validate Gadd45βas therapeutic target in multiple myeloma.

FIGS. 19A, 19B, and 19C show that the sh-RNA-mediated silencing ofeither MKK7 or Gadd45β does not affect cell-cycle distribution inmultiple myeloma cell lines. The representative lentivirus-infectedmultiple myeloma cell lines shown—that is ARH-77 (FIG. 19A), NCI-H929(FIG. 19B), and RPMI-8226 (FIG. 19C)—are from the same experimentexhibited in FIGS. 18A, 18B and 18 C. The cell cycle analyses shown inFIGS. 19A, 19B, and 19C were performed by the use of PI nuclear stainingassays, carried out as described in Example 8 (see also FIGS. 18A, B andC). Differently from the data shown in FIGS. 18A, B and C (in which PIstaining profiles are represented in a logarithmic scale, whichhighlights apoptosis), PI staining (that is FL2-A) in this Figures isrepresented in a linear scale, which highlights cell-cycle distribution.The percentages of multiple myeloma cells in the different phases of thecell cycle (that is G₁, S, and G₂/M) are depicted in the histograms.Cell-cycle analyses could not be performed with Gadd45β-specific sh-RNAsin the case of the ARH-77 (FIG. 19A) and NCI-H929 (FIG. 19B) multiplemyeloma cell lines, due to the induction of massive apoptosis afterexpression of these sh-RNAs (see FIGS. 18A and 18B). Nevertheless, as itcan be seen in FIG. 19A, Gadd4513 down-regulation had not effect oncell-cycle distribution in Z-DTP-resistant cell line, RPMI-8229.

Example 12 The Downregulation of MKK7 Expression Renders NormallySensitive Multiple Myeloma Cell Lines Completely Refractory toZ-DTP-Induced Killing

Materials and Methods

To assess the target specificity of Z-/mDTPs for the Gadd45β/MKK7complex, we investigated the effects of down-regulating the expressionof MKK7 on the sensitivity of susceptible multiple myeloma cell lines toZ-/mDTP-induced killing (FIGS. 20A, 20B, and 20C). To this end, weinfected the representative multiple myeloma cell line, ARH-77, withlentiviruses expressing MKK7-specific sh-RNAs, which result in thesilencing of the MKK7 gene, or of control non-specific sh-RNAs. The DNAsequences encoding the targeting small hairpin (sh)-RNAs are listed inTable VII. The MKK7-targeting sh-RNA sequences and the non-specificcontrol sequences were introduced between the BamHI and HpaI restrictionsites of the lentiviral vector, LentiLox3.7, as described in Example 11(see the reference by Yang et all 2006 PNAS 103, 10397-10402. Theproduction of hig-titer lentiviral preparation in HEK-293T cells wereperformed using essentially the same conditions described in thereference by Yang et all 2006 PNAS 103, 10397-10402. For introduction ofthe MKK7-targeting and the non-specific control sh-RNA sequences, therepresentative Z-DTP-sensitive multiple myeloma cell line, ARH-77, wasinfected with LentiLox3.7 lentiviruses expressing either MKK7-specificsh-RNAs (sh-MKK7) or non-specific sh-RNAs (sh-NS), as reported inpublished protocols essentially as described in the reference by Yang etall 2006 PNAS 103, 10397-10402. 5 days after infection, eGFP ARH-77cells were sorted using a BD FACSAria™ II cell sorter. Then, 10 daysafter cell sorting, lentivirus-infected multiple myeloma ARH-77 cellswere treated with either Z-DTP1, Z-DTP2, mDTP3 or Z-NC for 72 hrs at 37°C., or were cultured under the same conditions in the absence of peptidetreatment, as described in Example 8. The treatments with Z-DTP1,Z-DTP2, mDTP3 and Z-NC were carried out at the following final peptideconcentrations: 0.01 μM, 0.03 μM, 0.1 μM, 0.3 μM, 1 μM, 3 μM, and 10 μM.After these treatments, ARH-77 survival/proliferation was determined byperforming [³H]thymidine incorporation assays as described in Examples 6and 8. The results from these experiments were expressed as thepercentages of survival/proliferation (i.e. c.p.m.) observed inlentivirus-infected multiple myeloma cells treated with Z-DTP1, Z-DTP2,mDTP3 or Z-NC relative to the survival/proliferation of the respectivelentivirus-infected cells in the absence of peptide treatment. The meanconcentrations of Z-DTP1, Z-DTP2, mDTP3, and Z-DNC resulting in 50%(IC₅₀) inhibition of cell survival/proliferation were determined byperforming [³H]thymidine incorporation assays and were calculated asdescribed in Example 6. The results from these experiments are shown inFIGS. 20A, 20B, and 20C.

Results

FIGS. 20A, 20B, and 20C show that the sh-RNA-mediated silencing of MKK7renders the representative Z-/mDTP-sensitive cell line, ARH-77,completely resistant to Z-/mDTP-induced killing. The [³H]thymidineincorporation assays depicted in these Figures show the IC₅₀s ofD-isomer negative control tetrapeptide (Z-DNC) (FIGS. 20A, 20B, and20C), Z-DTP1 (FIG. 20A), Z-DTP2 (FIG. 20B), and mDTP3 (FIG. 20C) inARH-77 multiple myeloma cells expressing either MKK7-specific (sh-MKK7)or non-specific sh-RNAs (sh-NS). Treatments of ARH-77 cells were carriedout with different concentrations of these peptides and cellviability/proliferation analyzed by [³H]thymidine incorporation assaysafter 3 days. It can be seen that sh-NS-expressing ARH-77 cells arehighly sensitive to Z-/mDTP-induced killing—shown by the IC₅₀ values of1.4 μM (Z-DTP1; FIG. 20A), 302 nM (Z-DTP2; FIG. 20B), and 303 nM (mDTP3;FIG. 20C)—similar to what is seen in the uninfected, parental ARH-77cells (see Table IV). In striking contrast, however, sh-MKK7-expressingARH-77 cells became completely resistant to Z-/mDTP-inducedkilling—shown by the IC₅₀ values >10 μM of Z-DTP1, Z-DTP2, andmDTP3—similar to what is seen in Z-DNC-treated ARH-77 cells (FIGS. 20A,20B, and 20C). IC₅₀s were calculated as described in Example 6, usingincreasing concentrations of Z-DNC (FIGS. 20A, 20B, and 20C), Z-DTP1(FIG. 20A), Z-DTP2 (FIG. 20B), and mDTP3 (FIG. 20C), ranging from 0.01to 10 μM. Reported in the graphs are the percentages of the counts perminute (c.p.m.), a measure of cell survival/proliferation, obtained withtreated cells relative to the c.p.m. values obtained with untreatedcells. Similar data were obtained with additional Z-/mDTP-sensitivemultiple myeloma cell lines, including the U266, KMS-11, and KMS-12 celllines (data not shown). These data (i.e. the loss of Z-/mDTP sensitivityin susceptible multiple myeloma cell by the silencing of MKK7), togetherwith the data shown in FIG. 12 (i.e. the strong correlation betweenGadd45β expression and cancer cell sensitivity to Z-DTP-inducedkilling), conclusively demonstrate the very high target specificity ofZ-/mDTPs for the Gadd45β/MKK7 complex in multiple myeloma cells.

Example 13 Z-DTPs Retain Strong and Specific Cytotoxic Activity inPrimary Multiple Myeloma Cells from Patients

Materials and Methods

To confirm that Z-/mDTPs retain cytotoxic activity in primary multiplemyeloma cells, we examined the effects of Z-DTP1 and Z-DTP2 on thesurvival of multiple myeloma cells isolated from patients with aclinical diagnosis of multiple myeloma. To this end, multiple myelomacells were purified from bone marrow (BM) aspirates of multiple myelomapatients by negative selection, using CD138-conjugated magnetic beads,essentially as described in the reference by Hideshima T. et all 2006,Blood 107: 4053-4062. The purity of multiple myeloma cells was confirmedby flow cytometric, using and CD138 and anti-CD45 antibodies, alsoessentially in accordance with the procedure described in the referenceby Hideshima T. et all 2006, Blood 107: 4053-4062. Purified CD138⁺ BMcells were then cultured at a concentration of 4×10⁵ cells/ml in wellsof 96-well plates and treated with either 1 μM or 10 μM of Z-DTP1,Z-DTP2 or Z-DNC for 48 hrs. Cell viability was measured by cell countingusing trypan blue exclusion assays (FIGS. 14A, 14B, 14C, 14D, and 14E).

In order to determine the in vitro therapeutic index of Z-/mDTPs,viability and proliferation assays were also performed with primaryuntransformed cells of both human and mouse origin, after treatment witheither 10 μM or 100 μM of Z-DTP1, Z-DTP2 and Z-DNC. To this end, bonemarrow stromal cells (BMSCs) peripheral blood mononuclear cells (PBMNCs)and mesenkymal stem cells (MSCs) were purified from healthy individualsafter Ficoll-Hypaque density separation, in accordance with theprotocols reported in the reference by Piva R. et all 2008 Blood 111:2765-2775). BMSCs, PBMNCs, and MSCs cells were then treated for thetimes indicated and with the peptide concentrations specified in FIGS.15A and 15B. To further establish the specificity of the cytotoxicactivity of Z-/mDTPs for cancer cells, we also used primary B and Tlymphocytes purified from the spleen and lymph nodes of mice,respectively, essentially as described in the reference by Shirakawa etal 2010 Cell Mol immunology 1-12. B and T cells were then activated bysimulation with 1 ng/mL of LPS for 16 hrs and subsequently treated with100 μM of Z-DTP1, Z-DTP2 or Z-DNC for 72 hrs as shown in FIG. 15B.

Results

FIGS. 14A, 14B, 14C, 14D, and 14E show that Z-DTP1 and Z-DTP2, but notZ-DNC, exhibit strong cytotoxic activity in primary multiple myelomacells isolated from 5 representative patients. Each panel depicts thedata obtained with multiple myeloma cells from a different patient—thatis patient 1 (FIG. 14A), patient 2 (FIG. 14B), patient 3 (FIG. 14C),patient 4 (FIG. 14D), and patient 5 (FIG. 14E). Treatments with Z-DTP2,Z-DTP1 and Z-DNC were at the concentrations indicated (i.e. 1 μM or 10μM), for 48 hrs. Assays were performed using trypan blue exclusion.Values represent the percent of live cells after treatment with Z-DTP2,Z-DTP1 or Z-DNC relative to the viability of untreated control cells.Strong cytotoxic activity—comparable to that of Z-DTP2 and Z-DTP1—wasalso observed in primary myeloma cells from patients with mDTP3, undersimilar experimental conditions (data not shown). These findingsdemonstrate that Z-/mDTPs retain activity in primary multiple myelomacells and indicate that Z-/mDTP-based therapy can be used in patients totreat multiple myeloma.

FIGS. 15A and 15B show that Z-DTP1 and Z-DTP2 exhibit no toxicity tonormal primary cells of either mouse or human origin, even when used atvery high concentrations—that is 100 μM. The primary cells testedincluded normal bone marrow stromal cells (BMSCs) (FIG. 15A), peripheralblood mononuclear cells (PBMNCs) (FIG. 15A), and mesenkymal stem cells(MSCs) (FIG. 15B), isolated from multiple myeloma-free individuals, andpurified primary B and T lymphocytes isolated from mice (FIG. 15B).Treatments with Z-DTP2, Z-DTP1 and Z-DNC were at the concentrationsindicated, for either: 48 hrs (BMSCs, PBMNCs) (FIG. 15A), 72 hrs (murineB and T cells) (FIG. 15B), or 144 hrs MSCs (FIG. 15B). Assays wereperformed by using trypan blue exclusion (FIG. 15A) or [³H]thymidineincorporation (FIG. 15B). The data presented in FIGS. 14 and 15 indicatethat Z-DTPs have a high in vitro therapeutic indices (i.e. lack oftoxicity in normal cell versus a high toxicity in cancer cells). Indeed,Z-DTP1 and Z-DTP2, but not Z-DNC, show strong tumoricidal activity inmultiple myeloma cells from patients (FIG. 14), but exhibit no toxicityin primary normal cells from healthy individuals or mice (FIG. 15A),even when used at very high concentrations such as 100 μM (see FIG.15B). These data demonstrate that Z-DTPs do not have indiscriminatedcytotoxic effects in cells—rather their cytotoxic effects are specificfor cancer cells and/or cells featuring high levels of Gadd45βexpression or activity and/or constitutive high expression or activityof NF-κB.

The high activity of Z-/mDTPs in multiple myeloma and other cancercells, combined with their lack of toxicity in primary normal cells,including primary human BMSCs, MSCs, PBMNCs and mouse B and Tlymphocytes, even when used at high concentrations (ie 100 μM),demonstrate that the compounds of the invention have excellent in vitrotherapeutic indices (see FIG. 9, lack of toxicity in cell lines that donot depend on NF-κB for survival; FIG. 12, correlation between Gadd45βexpression and cancer cell sensitivity to Z-DTPs; FIGS. 14 and 15,killing assays in primary cells)—a key advantage of our invention overexisting therapies. The compounds of the invention also lack toxicity intumour cell lines such as T-cell leukemia, Burkitt's lymphoma and manyothers, which do not depend on NF-κB for survival (even when used at 100μM; see FIG. 9), showing that their activity has inherent specificityfor cells with constitutively active NF-κB. Furthermore, in a largepanel of tumour cell lines of different tissues of origin, there is ahighly statistically significant correlation between levels of Gadd45βexpression and sensitivity to Z-/mDTP-induced killing (p<0.01; FIG. 12),thereby establishing the high specificity of the Z-/mDTPs' cytotoxicaction for Gadd45β. Crucially, the sh-RNA-mediated mediateddown-regulation of Gadd45β causes apoptosis in Z-/mDTP-sensitivemultiple myeloma cell lines (e.g. ARH-77 and NCIH929) with kineticssimilar to those seen with Z-/mDTPs, but not in Z-/mDTP-resistantmultiple myeloma cell lines (e.g. RPMI-8226), and the sh-RNA-mediatedmediated down-regulation of MKK7 causes results in a loss of sensitivityto Z-/mDTP-induced killing in susceptible multiple myeloma cell lines(e.g. ARH-77) (see FIG. 20). Together, our data show that the cytotoxicactivity of Z-/mDTPs is restricted to tumour cells featuringconstitutively active NF-κB and/or high levels of Gadd45β expression oractivity—Z-/mDTPs exhibit cytoxicity at nM levels in sensitive multiplemyeloma cell lines, but have no toxicity in resistant tumour lines thatdo not depend on NF-κB for survival or that exhibit low levels ofGadd45β expression, even when used at 100 μM. Moreover, in contrast tomice lacking core components of the IKK/NF-κB pathway, gadd45β^(−/−)mice are viable and seemingly healthy (Papa et all 2008 J Clin Invest118, 1911-1923), indicating that (unlike full proteasome/NF-κB blockade)complete Gadd45β inactivation is well tolerated in vivo (Papa et all2008 J Clin Invest 118, 1911-1923). Together, these findings indicatethat Z-/mDTP-based therapy will be safe and specific (see FIGS. 9 and15, lack of toxicity in NF-κB-independent tumour cell lines and normalprimary cells; FIG. 12, correlation between Gadd45β expression andcancer cell sensitivity to Z-/mDTP-induced killing; FIG. 14,Z-/mDTP-induced killing of primary multiple myeloma cells).

Proteasome inhibitors (PIs), such bortezomib, and other multiple myelomatherapies also kill multiple myeloma cells by activating JNK (Chauhan etal 2008 Blood 111, 1654-1664), but cannot cure due to low therapeuticindices (Lauback et al 2009 Leukemia 23, 2222-2232; Ludwing et al 2010Oncologist 15, 6-25 and www.cancecare.on.ca/). Targeting the discretefunctions of NF-κB in multiple myeloma survival via Gadd45β will enableto dissociate NF-κB's functions in immunity, inflammation and survival,so provide a safer, more specific therapy that can be tolerated at dosesrequired to cure. Z-/mDTPs define an entirely new class of therapeuticagents targeting a novel pathway in multiple myeloma, and potentiallyother cancers and diseases or disorder that depend on NF-κB forsurvival.

Example 14 Binding Properties of mDTP3 to Gadd45β and MKK7 Proteins inIsolation and as Part of a Gadd45β/MKK7 Complex

By way of example, binding experiments were performed with mDTP3 toGadd45β, the kinase domain of MKK7 (MKK7_(KD)) and to the Gadd45β/MKK7complex using the Surface Plasmon Resonance technique.

Materials and Methods

To determine how DTPs bind to the Gadd45β/MKK7 complex, experiments wereperformed with a Biacore3000 SPR instrument (GE Healthcare, Milan,Italy), using 4-channels CM5 sensorchips (GE Healthcare, Milan, Italy).Full length human Gadd45β was prepared and purified as described in thereference by Tornatore L., et al. (2008). J Mol Biol; 378:97-111. Theconstitutively active kinase domain of MKK7, spanning residues 101 to405, and carrying the S287D and T291D mutations (MKK7_(KD)), wasexpressed in E. Coli as a fusion protein of His6. The protein waspurified to homogeneity by two subsequent steps of affinitychromatography (Ni-NTA support) and gel filtration (Superdex G75), andthen characterized by SDS-PAGE, LC-MS to verify identity and purity, andby Circular Dichroism to assess folding.

MKK7_(KD) was immobilized on the Biacore sensorchip via classicalEDC/NHS coupling chemistry at pH 5 (protein pI, ˜9) at a flow rate of 5μL/min. An immobilization level of about 8000 Response Units wasachieved. Gadd45β, which is an intrinsically acidic protein with a pI ofabout 4.5, was immobilized at pH 3.5 (6000 RU immobilization levels) ona separate channel. The residual reactive groups on both the Gadd45β andMKK7_(KD) channels were finally inactivated by treatment withethanolamine. On another channel the same procedure of activation withEDC/NHS and inactivation with ethanolamine was performed. This channelwas used as reference and the signal deriving from it was considered asblank, and values were accordingly subtracted from the experimentalchannels detecting Gadd45β or MKK7_(KD) proteins to remove non-specificbinding to the chip surface. To determine whether the two proteins wereeffectively immobilized, we performed repeated injections of Gadd45β(20-200 nM) and MKK7_(KD) (1-25 nM) at increasing protein concentrations(3 min contact time; 60 μL). Regeneration was achieved using either 1MNaCl injections (1 min, MKK7_(KD)-derivatized channel) or 20 mM NaOH (30sec, Gadd45β-derivatized channel).

Increasing concentrations of the tripeptide mDTP3(Ac-D-Tyr-D-Arg-D-Phe-NH₂) were finally injected over the chip atconcentrations ranging between 1 nM and 10 μM. In a separate experiment,mDTP3 was injected during the dissociation phase of either Gadd45β fromimmobilized MKK7_(KD) or of MKK7_(KD) from immobilized Gadd45β. Theresults from these analyses are reported in FIGS. 21A, 21B, 21C, and21D.

Results

As it can be seen in FIG. 21A, the binding of Gadd45β to immobilizedMKK7_(KD) was very effective. Dose-response association and dissociationcurves were observed at all the concentration used. The dissociationconstant K_(D) of the Gadd45β/MKK7_(KD) interaction was estimated byaveraging the values calculated over each of the different curves anddetermined to be 4.0±0.7 nM (see FIG. 21A). Similarly, repeatedinjections of MKK7_(KD) on the Gadd45β channel provided dose-responseassociation and dissociation curves (FIG. 21B) from which a K_(D) of3.4±0.6 nM was derived.

To determine whether mDTP3 binds to MKK7_(KD) and/or to Gadd45β, samplesof the peptide (i.e. mDTP3) were injected over the Gadd45β andMKK7_(KD)-derivatized channels. Surprisingly, as it can be seen in FIG.21C, the data show that this peptide does not bind to either Gadd45β orMKK7_(KD) in isolation. Strikingly, however, when mDTP3 was injectedduring the dissociation phase of Gadd45β from MKK7_(KD) (FIG. 21D) orthe dissociation phase of MKK_(KD) from Gadd45β (data not shown),binding was observed and dose-response association and dissociationcurves could be recorded. FIG. 21D shows that when mDTP3 was injected atthe low concentrations of either 10 nM or 100 nM, this peptide induced arapid dissociation of the Gadd45β/MKK7_(KD) complex. As it can also beseen, Gadd45β/MKK7_(KD) complex formation was rapidly recovered afterthe peptide was washed away. FIG. 21D also shows that when mDTP3 wasinjected at higher concentrations (e.g. 1 μM), dose-response binding anddissociation curves were recorded, indicating that mDTP3 was binding toeither Gadd45β and/or to MKK7_(KD) or to a complex of the two proteins.Together, these data demonstrate that mDTP3 is unable to bind to eitherGadd45β or MKK7_(KD) in isolation, even when used at highconcentrations, rather its binding to either Gadd45β, MKK7_(KD), or asurface created by interaction of the two proteins requires formation ofa Gadd45β/MKK7 complex. These data are important, as they show that ourtherapeutic target is the interface between two proteins (i.e. Gadd45βand MKK7)—which provides potential for high target selectivity in cells,a key advantage of our invention over existing therapies.

Example 15 In Vivo Pharmacokinetical (DMPK) Profiles of Z-DTP2 and mDTP3

To assess the suitability of Z-DTP2 and mDTP3 for therapeutic use invivo, we performed pharmacokinetical analyses in mice.

Materials and Methods

Mouse Pharmacokinetics Study:

Protocol Summary:

Z-DTP2 and Z-mDTP3 were administered intravenously to mice. Bloodsamples were collected at up to 7 time points after intravenous (i.v.)injection of the compounds over 8 hrs, and plasma was analysed byLC-MS/MS to determine the concentration of the compounds at each timepoint.

Experimental Procedure:

Three male CD1 mice, 25-30 grams each, were dosed per administrationroute per time-point, per compound. The test compound was administeredintravenously (at a typical dose level of 10 mg of compound per kg ofbody weight). Animals were given free access to food throughout thestudy.

At the following time points, the animals were anaesthetised, blood wascollected in heparinised tubes, and the animals were sacrificed:

-   -   i.v. dosing: 0.08, 0.25, 0.5, 1, 2, 4 and 8 hrs post-dosing        Sample Preparation:

Blood samples were centrifuged to obtain the plasma, which was thentransferred to a separate labelled container. Aliquots from theindividual time points for the three animals were analysed singly.Proteins were precipitated by adding three volumes of methanol andcentrifuging for 30 min at 4° C. Aliquots of 100 μl of the resultingsupernatants were diluted with 200 μl of HPLC grade water in wells of a96-well plate.

Quantitative Analysis:

Standard curves were prepared in blank plasma matrices and treated in anidentical manner to the samples. The plasma samples were quantified byLC-MS/MS, and the concentration of each compound in the plasma werereported in μg/mL.

Pharmacokinetic Analysis:

Pharmacokinetic parameters were calculated employing non-compartmentalmodel analysis, as described in the web sitehttp://www.pharsight.com/main.php

Bioanalysis:

Protocol Summary:

The test compound concentration in plasma samples was measured byLC-MS/MS. The data were quantified using a five-point standard curveover a range of 3-3000 ng/mL.

Experimental Procedure:

Proteins were precipitated from 50 μL aliquots of the individual plasmasamples by adding 150 μL methanol. Following protein precipitation,plasma samples were centrifuged for 30 min at 4° C. Aliquots of 100 μLof the resulting supernatant were diluted with 200 μL of HPLC gradewater in a 96 well plate. The test compound was then quantified byLC-MS/MS from a five-point standard curve prepared by spiking plasmawith varying concentrations of test compound dissolved in DMSO over afinal concentration range of 3-3000 ng/mL (final DMSO concentration 1%)and treated in an identical manner to the test samples as describedabove.

Results

Pharmacokinetical studies in male CD1 mice show that both Z-DTP2 andmDTP3 have in vivo DMPK profiles suitable for administration viaintravenous (i.v.) infusion (see Tables VIII, IX [A], and IX [B]), inthe absence of acute toxicity in mice. Table VIII reports the values ofthe most important in vivo pharmacokinetical parameters obtained withZ-DTP2 and mDTP3, including half-life in plasma (T_(1/2)), steady state(Vss) and terminal (Vβ) Volumes of distribution, and total clearance(tot CL), area under the plasma concentration versus time curve (AUC),and concentration at time point 0 (C₀). Values were calculated from thedata of plasma concentration versus time curves based on thenon-compartmental and compartimental methods of analysis (Groulx A. 2006ScianNew 9: 1-5 and DiStefano 3rd 1982 Am J Physiol Regul Integr CompPhysiol 243: 1-6) (data not shown). Each parameter shown represents theaverage of experimental values obtained from three different pools ofmale CD1 mice following a single intravenous (i.v.) injection of thecompounds at a dose of 10 mg per kg of body weight. Three male CD1 mice(25-30 gr of body weight) were dosed via i.v. administration of eitherZ-DTP2 or mDTP3. Blood samples were collected at 7 time points as shown(i.e. at 0.08, 0.25, 0.5, 1, 2, 4 and 8 hrs after injection) and theplasma was analysed by liquid chromatography mass spectrometry (LC-MS)to determinate the blood concentrations of the two compounds at eachtime point. The plots of the plasma concentration versus time profilewere extrapolated for both Z-DTP2 and mDTP3. The results show thatZ-DTP2 and mDTP3 both follow a multiphasic disposition after intravenousinjection (data not shown). Indeed, the concentration-versus-time curvesof the intravenously administered compounds display a distinctbio-exponential profile with a steep initial distribution phase and along terminal T_(1/2) (data not shown).

The main pharmacokinetical parameters extrapolated from the data ofplasma concentration-time curves (i.e. C₀, AUC to last, T½, Vβ, Vss, andCL) are crucial for calculating the dosing levels and regiment ofadministration, required to achieve the desired systemic steady stateconcentrations of a drug (i.e. the therapeutic systemic concentrations).As it can be seen in Table VIII, Z-DTP2 and mDTP3 exhibit half-lives invivo of approximately 2 hrs and of approximately 1 hr and 20 min,respectively.

Interestingly, Z-DTP2 and mDTP3 both show an initial distributivehalf-life of approximately 5 min, which could suggest rapidtissue/cellular uptake, but alternatively could suggest binding toplasma proteins. Most importantly, both compounds exhibit very slowelimination from the tissues, which is reflected by a terminal half-lifeof approximately 8 hrs (Table VIII and data not shown)(http://www.pharsight.com/main.php andhttp://www.meds.com/leukemia/idamycin/adriamycin.html and Kupperman etal 2010 Cancer Res 70 1970-1980). The data also show that Z-DTP2 andmDTP3 both follow a general linear pharmacokinetic system (Berezhkovskiy(2007) J Pharm Sci. 96, 1638-52), as indicated by the finding that theirvalues of total volume distribution are higher then those of steadystate volume distribution (i.e. Vβ>Vss).

Both the terminal and steady state volume distributions as well as theterminal half-lives of the two compounds synergistically contribute toestablish the quantity of drug required in the body for a constant rateof infusion.

Importantly, Z-DTP2 and mDTP3 show values of total clearance in therange of 66 to 90 mL/min/kg and of 22 to 27 mL/min/kg, respectively,suggesting slow metabolic and biliary excretion rates for both compounds(Table VIII and data not shown).

Tables IX [A] and IX [B] show the predicted dosing for in vivoadministration of Z-DTP2 and mDTP3, respectively, required to achievesystemic therapeutic concentrations of the two compounds. The valuesreport the dosing expressed in mg/hr required to obtained the desiredsteady state plasma concentrations of 1, 5 or 10 μM for either Z-DTP(Table IX [A]) or mDTP3 (Table IX [B]). Significantly, despite having acomparable half-life as well as a comparable terminal half-life toZ-DTP2, mDTP3 exhibits a total clearance value that is 3 times lowerthen that of Z-DTP2 (Table VIII and data not shown). Of note, even asmall difference in this crucial pharmacokinetical parameter maysignificantly affect the dosing size and regimen required to achieve thedesired steady state plasma concentration of a compound, as seen withthe difference in the dosings predicted for Z-DTP2 and mDTP3 (Tables IX[A] and IX [B], respectively). Indeed, Tables IX [A] and IX [B](modelling analyses) show that in order to achieve a steady state plasmaconcentration of 1, 5, or 10 μM, the dosing required for mDTP3 issignificantly lower than that required for Z-DTP2. Thus, based on thesepharmacokinetic results and on the IC₅₀ values determined for the twocompounds in multiple myeloma cell lines (see Table IV) in order toachieve a steady state plasma concentration of up to 10 μM it will benecessary to administer Z-DTP2 and mDTP3 via continuous i.v. infusion ata rate of 0.976 mg/hr and 0.218 mg/hr, respectively (Tables IX [A] andIX [B]).

Of note, Z-/mDTP synthesis, is concise and straightforward, hencecost-effective even for chronic use. Thus, even with low T_(1/2),Z-/mDTP therapy by infusion will be acceptable in hospitalized patientsalready on chemotherapy. The compounds of the invention are also highlysoluble and have high specificity and good safety profiles, so can bedelivered at high doses, in low volumes to maximize therapeutic effects,as successfully exploited by existing peptide therapies.

Tables

TABLE I Initial Elisa Screening SEQ % Inhibition of Amino acid sequenceID Gadd45β-MKK7 (single-letter code) NO: MW bindingFmoc(βAla)₂-QX₂X₃X₄-NH₂ 156 — 0 Fmoc(βAla)₂-SX₂X₃X₄-NH₂ 157 — 4Fmoc(βAla)₂-RX₂X₃X₄-NH₂ 158 — 45 Fmoc(βAla)₂-AX₂X₃X₄-NH₂ 159 — 56Fmoc(βAla)₂-YX₂X₃X₄-NH₂ 160 — 100 Fmoc(βAla)₂-PX₂X₃X₄-NH₂ 161 — 48Fmoc(βAla)₂-MX₂X₃X₄-NH₂ 162 — 36 Fmoc(βAla)₂-CX₂X₃X₄-NH₂ 163 — 49Fmoc(βAla)₂-FX₂X₃X₄-NH₂ 164 — 58 Fmoc(βAla)₂-LX₂X₃X₄-NH₂ 166 — 55Fmoc(βAla)₂-HX₂X₃X₄-NH₂ 167 — 56 Fmoc(βAla)₂-DX₂X₃X₄-NH₂ 168 — 55Fmoc(βAla)₂-YDHQ-NH₂ 165 — 26 Fmoc(βAla)₂-YSX₃X₄-NH₂ 169 — 16Fmoc(βAla)₂-YRX₃X₄-NH₂ 170 — 28 Fmoc(βAla)₂-YAX₃X₄-NH₂ 171 — 20Fmoc(βAla)₂-YYX₃X₄-NH₂ 172 — 52 Fmoc(βAla)₂-YPX₃X₄-NH₂ 173 — 42Fmoc(βAla)₂-YMX₃X₄-NH₂ 174 — 54 Fmoc(βAla)₂-YCX₃X₄-NH₂ 175 — 27Fmoc(βAla)₂-YFX₃X₄-NH₂ 176 — 39 Fmoc(βAla)₂-YLX₃X₄-NH₂ 177 — 52Fmoc(βAla)₂-YHX₃X₄-NH₂ 178 — 53 Fmoc(βAla)₂-YDX₃X₄-NH₂ 179 — 96Fmoc(βAla)₂-YDQX₄-NH₂ 180 — 19 Fmoc(βAla)₂-YDSX₄-NH₂ 181 — 11Fmoc(βAla)₂-YDRX₄-NH₂ 182 — 93 Fmoc(βAla)₂-YDAX₄-NH₂ 183 — 0Fmoc(βAla)₂-YDYX₄-NH₂ 184 — 25 Fmoc(βAla)₂-YDPX₄-NH₂ 185 — 25Fmoc(βAla)₂-YDMX₄-NH₂ 186 — 13 Fmoc(βAla)₂-YDCX₄-NH₂ 187 — 6Fmoc(βAla)₂-YDFX₄-NH₂ 188 — 37 Fmoc(βAla)₂-YDLX₄-NH₂ 189 — 30Fmoc(βAla)₂-YDHX₄-NH₂ 190 — 99 Fmoc(βAla)₂-YDDX₄-NH₂ 191 — 37Fmoc(βAla)₂-YDHQ-NH₂ 192 925.94 0 Fmoc(βAla)₂-YDHS-NH₂ 193 884.88 0Fmoc(βAla)₂-YDHR-NH₂ 194 953.99 2 Fmoc(βAla)₂-YDHA-NH₂ 195 868.89 15Fmoc(βAla)₂-YDHY-NH₂ 196 960.98 63 Fmoc(βAla)₂-YDHP-NH₂ 197 894.92 16Fmoc(βAla)₂-YDHM-NH₂ 198 928.99 14 Fmoc(βAla)₂-YDHC-NH₂ 199 900.95 44Fmoc(βAla)₂-YDHF-NH₂ 200 944.98 99 (Fmoc-LTP1) Fmoc(βAla)₂-YDHL-NH₂ 201910.96 33 Fmoc(βAla)₂-YDHH-NH₂ 202 934.94 40 Fmoc(βAla)₂-YDHD-NH₂ 203912.89 0

TABLE II Modified pure peptides Amino acid sequence SEQ ID % Inhibitionof Gadd45β- (single-letter code) NO: MW MKK7 binding Ac-YDHF-NH₂ 61 62194 (Ac-LTP1) Ac-YEHF-NH₂ 41 636 52 Ac-WDHF-NH₂ 85 645 28 Ac-WEHF-NH₂ 43659 36 Ac-YDRF-NH₂ 42 640 45 Ac-YDKF-NH₂ 208 612 35 Ac-YEKF-NH₂ 40 62664 Ac-YERF-NH₂ 27 654 93 (Ac-LTP2) Ac-WEKF-NH₂ 44 649 65 Ac-WERF-NH₂ 86678 29 Ac-WDKF-NH₂ 46 659 26 Ac-WDRF-NH₂ 87 663 46 Ac-YDHW-NH₂ 88 661 58Ac-YEHW-NH₂ 90 675 64 Ac-WDHW-NH₂ 91 683.7 50 Ac-WEHW-NH₂ 92 698 75Ac-YDRW-NH₂ 93 679 43 Ac-YDKW-NH₂ 94 622 23 Ac-YEKW-NH₂ 95 666 27Ac-YERW-NH₂ 96 694 59 Ac-WEKW-NH₂ 97 689 65 Ac-WERW-NH₂ 98 717 69Ac-WDKW-NH₂ 99 674 69 Ac-WDRW-NH₂ 100 702 93 Ac-YDHQ-NH₂ 101 602 99

TABLE III Modified peptides Amino acid sequence SEQ ID % Inhibition ofGadd45β- (single-letter code) NO: MW MKK7 binding Ac-YEHF-NH₂ 41 636 23Ac-YDRF-NH₂ 42 642 19 Ac-AERF-NH₂ 102 563 7 Ac-YARF-NH₂ 103 597 17Ac-YEAF-NH₂ 104 570 13 Ac-YERA-NH₂ 105 579 28 Ac-PERF-NH₂ 106 589 24Ac-YPRF-NH2 108 623 13 Ac-YEPF-NH₂ 107 596 13 Ac-YERP-NH₂ 109 605 0Z-YERF-NH₂ 60 747 78 (Z-LTP2) Z-YDHF-NH₂ 61 714 84 (Z-LTP1) Z-YDHQ-NH₂101 695 13 Z-YD(OMe)HF-NH₂ 62 729 84 Z-YD(OMe)HQ-NH₂ 111 710 32CI-Z-YERF-NH₂ 112 783 38 2CI-Z-YDHF-NH₂ 113 749 38 2CI-Z-YDHQ-NH₂ 114730 40 Myr-YERF-NH₂ 70 823 37 Myr-YDHF-NH₂ 71 790 26 Myr-YDHQ-NH₂ 771 28Benzoic acid-YERF-NH₂ 112 718 24 Benzoic acid-YDHF-NH₂ 684 0 Benzoicacid-YDHQ-NH₂ 110 665 1 3-OH—4MeO-Benzoid acid- 63 763 86 YERF-NH₂3-OH—4MeO-Benzoid acid- 64 732 81 YDHF-NH₂ 3-OH—4MeO-Benzoid acid- 115713 7 YDHQ-NH₂ Fmoc-YERF-NH₂ 66 835 60 Fmoc-YDHF-NH₂ 69 802 58Fmoc-YDFQ-NH₂ 116 783 19 Ac-YERFLys(Z)-NH₂ 204 917 6 Ac-YDHFLys(Z)-NH₂205 884 8 Ac-YDHQLys(Z)-NH₂ 206 865 2

TABLE IV IC₅₀s of Z-DTP1 IC₅₀s of Z-DTP2 MM cell lines Day 1 Day 3 Day 6Day 1 Day 3 Day 6 KMS12 6.0 μM 537 nM 316 nM 1.3 μM 144 nM 67 nm KMS114.26 μM 51.3 nM 10.1 nM 2.88 μM 25.7 nM 10 nM ARH-77 >10 μM 950 nM 2.2μM >10 μM 218 nM 1.2 μM NCI 5.25 μM 4.07 μM 776 nM 5.25 μM 2.35 μM 501nM U266 6.3 μM 81.3 nM 40.7 nM 6.02 μM 67.7 nM 40.7 nM JJN3 10 μM 1.1 μM350 nM 10 μM 1 μM 223 nM KMS18 7.9 μM 6.2 μM 3.7 μM 9.8 μM 3.4 μM 3.0 μMKMS27 >10 μM >10 μM 4.9 μM >10 μM 1.6 μM 4.5 μM

TABLE V IC₅₀ IC₅₀ [H³] thymidine [H³] thymidine incorporation assay inincorporation assay in Amino acid sequence IC₅₀ KMS12 KMS11(three-letter code) ELISA Day 1 Day 3 Day 6 Day 1 Day 3 Day 6 1Z-Tyr-Asp-His-Phe-NH₂ 220 pM 6.0 μM 537 nM 316 nM 4.26 μM 51.3 nM 10.1nM (Z-DTP1) SEQ ID NO: 61 2 X₁-Asp-His-Y₁ >1 nM >10 μM >10 μM >10 μM >10μM >10 μM >10 μM 3 X₁-Asp-His-Y₂ >1 nM >10 μM >10 μM >10 μM >10 μM >10μM >10 μM 4 X₁-Asp-His-Y₃ 500 pM >10 μM >10 μM >10 μM >10 μM >10 μM >10μM 5 X₂-Asp-His-Y₁ 316 pM >10 μM >10 μM >10 μM >10 μM >10 μM >10 μM 6X₂-Asp-His-Y₂ 250 pM >10 μM >10 μM >10 μM >10 μM >10 μM >10 μM 7X₂-Asp-His-Y₃ 100 pM 8.5 μM 380 nM 199 nM 2.5 μM 1.17 μM 549 nM (mDTP4)8 Z-Tyr-Asp-Phe-NH₂ 162 pM — — — >10 μM >10 μM >10 μM 9Z-Tyr-Glu-Arg-Phe-NH₂ 190 pM 1.3 μM 141 nM 66 nM 2.9 μM 25.7 nM l0 nM(Z-DTP2) SEQ ID NO: 60 10 X₁-Glu-Arg-Y₁ 500 pM — — — >10 μM >10 μM >10μM 11 X₁-Glu-Arg-Y₂ 500 pM — — — >10 μM >10 μM >10 μM 12 X₁-Glu-Arg-Y₃301 pM — — — >10 μM >10 μM >10 μM 13 X₂-Glu-Arg-Y₁ >1 nM — — — >10μM >10 μM >10 μM 14 X₂-Glu-Arg-Y₂ >1 nM — — — >10 μM >10 μM >10 μM 15X₂-Glu-Arg-Y₃ 100 pM 6.0 μM 301 nM 436 nM 2.8 μM 562 nM 263 nM (mDTP1)16 Z-Tyr-Glu-Phe-NH₂ 158 pM 6.5 μM 3 μM 288 nM (mDTP2) 17Ac-Tyr-Arg-Phe-NH₂ 157 pM 354 nM 81 nM 16 nM 1 μM 89 μM 25 nM (mDTP3) 18Ac-Tyr-Tyr-Arg-Phe-NH₂ >5 nM — — — — — — SEQ ID NO: 117 19Z-Tyr-Arg-Phe-NH₂ 100 pM 354 nM 81 nM 20 nM — — — 20Ac-Cha-Arg-Phe-NH₂ >5 nM — — — — — — 21 Ac-Tyr-Arg-Cha >5 nM — — — — — —22 Z-Tyr-Tyr-Glu-Arg-Phe- >5 nM — — — — — — NH₂ SEQ ID NO: 118 23Ac-Tyr-Gln-Arg-Phe-NH₂ 5 nM — — — >10 μM >10 μM >10 μM (Elisa) SEQ IDNO: 57 Z-Tyr-Gln-Arg-Phe-NH₂ ([H³] assay) SEQ ID NO: 121 24Ac-Tyr-Met-Arg-Phe-NH₂ >10 nM — — — >10 μM >10 μM >10 μM (Elisa) SEQ IDNO: 59 Z-Tyr-Met-Arg-Phe-NH₂ ([H³] assay) SEQ ID NO: 122 25Ac-Tyr-Asn-Arg-Phe-NH₂ 10 nM — — — >10 μM >10 μM >10 μM (Elisa) SEQ IDNO: 123 Z-Tyr-Asn-Arg-Phe-NH₂ ([H³] assay) SEQ ID NO: 124 26Ac-Tyr-Leu-Arg-Phe-NH₂ 5 nM — — — >10 μM >10 μM >10 μM (Elisa) SEQ IDNO: 125 Z-Tyr-Leu-Arg-Phe-NH₂ ([H³] assay) SEQ ID NO: 126 27Ac-Tyr-Gln-Phe-NH₂ >10 nM — — — >10 μM >10 μM >10 μM (Elisa)Z-Tyr-Gln-Phe-NH₂ ([H³] assay) 28 Ac-Tyr-Leu-Phe-NH₂ 1.8 nM — — — >10μM >10 μM >10 μM (Elisa) Z-Tyr-Leu-Phe-NH₂ ([H³] assay) 29Ac-Tyr-Asn-Phe-NH₂ 1.9 nM — — — >10 μM >10 μM >10 μM (Elisa)Z-Tyr-Asn-Phe-NH₂ ([H³] assay) 30 AC-Tyr-Met-Phe-NH₂ >10 nM — — — >10μM >10 μM >10 μM (Elisa) Z-Tyr-Met-Phe-NH₂ ([H³] assay) 31Ac-Tyr-Gln-Phe-NH₂ >10 nM — — — >10 μM >10 μM >10 μM (Elisa)ZTyr-Gln-Phe-NH₂ ([H³] assay) 32 Z-Tyr-Asp-His-Gln-NH₂ >10 nM — — — — —— SEQ ID NO: 127 33 Z-Tyr-Tyr-Asp-His Gln- >10 nM — — — — — — NH₂ SEQ IDNO: 128

TABLE VI Compounds Amino acid sequence IC₅₀ ID (single-letter code)Elisa A1 Ac-YF-NH₂ >100 nM A1 bis Ac-FF-NH₂ >100 nM A3Ac-YbetaAla-F-NH₂ >100 nM A6 Ac-Y-eCaprioic-F-NH₂ >100 nM A7Ac-YY-NH₂ >100 nM A8 Ac-FY-NH₂ >100 nM A9 Ac-FRF-NH₂ >100 nM B2Ac-YKF-NH₂ 0.851 nM  B13 Ac-YPF-NH₂ 0.645 nM  B16 Ac-YHF-NH₂ 0.690 nM B16 bis H-YHF-NH₂ 0.645 nM  O1 Ac-FRY-NH₂ >100 nM O3 Ac-YRY-NH₂ 0.758nM  O5 H-FHY-NH₂ >100 nM O6 H-YRY-NH₂ 0.750 nM  O5 bis Ac-FHY-NH₂ >100nM O7 H-FRF-NH₂ >100 nM O8 H-FRY-NH₂ >100 nM

TABLE VII RNA interference Targeting Gene Sequences Forward Reverse ns-1CAGTCGCGTTTGCG TCAGTCGCGTTTGCGAC TCGAGAAAAAACAGTCGC ACTGGTGGTTCAAGAGACCAG GTTTGCGACTGGTCTCTTG SEQ ID NO: 129 TCGCAAACGCGACTGTAACCAGTCGCAAACGCGA TTTTTC CTGA SEQ ID NO: 130 SEQ ID NO: 131 ns-2AAGTATGGTGAGC TAAGTATGGTGAGCAC TCGAGAAAAAAAAGTATG ACGCGTGCGTTTCAAGAGAACG GTGAGCACGCGTTCTCTTG SEQ ID NO: 132 CGTGCTCACCATACTTTAAACGCGTGCTCACCATA TTTTTC CTTA SEQ ID NO: 133 SEQ ID NO: 134 Gadd45β-1CCAAGTTGATGAAT TCCACTGTCTTCCCTTC GAAAAAACCAAGTTGATG GTGGACTATTCAAGAGATAGG AATGTGGATCTCTTGAATC SEQ ID NO: 135 AAGGGAAGACAGTGGTCACATTCATCAACTTGGA TTTTTC SEQ ID NO: 137 SEQ ID NO: 136 Gadd45β-2CAGAAGATGCAGA TCAGAAGATGCAGACG TCGAGAAAAAACAGAAGA CGGTGAGTGATTCAAGAGATCA TGCAGACGGTGATCTCTTG SEQ ID NO: 138 CCGTCTGCATCTTCTGTAATCACCGTCTGCATCTTC TTTTTC TGA SEQ ID NO: 139 SEQ ID NO: 140 Gadd45β-3CAAATCCACTTCAC TCAAATCCACTTCACGC TCGAGAAAAAACAAATCC GCTCATCATTCAAGAGATGAG ACTTCACGCTCATCTCTTG SEQ ID NO: 141 CGTGAAGTGGATTTGTTAATGAGCGTGAAGTGGAT TTTTC TTGA SEQ ID NO: 142 SEQ ID NO: 143 MKK7-1GATCACAGGAAGA TGATCACAGGAAGAGA TCGAGAAAAAAGATCACA GACCAA CCAATTCAAGAGAGGAAGAGACCAA SEQ ID NO: 144 TTGGTCTCTTCCTGTGA TCTCTTGAATTGGTCTCTTTCTTTTTTC CCTGTGATCA SEQ ID NO: 145 SEQ ID NO: 146 MKK7-2 GCATTGAGATTGACTGCATTGAGATTGACC TCGAGAAAAAAGCATTGA CAGAA AGAATTCAAGAGATTC GATTGACCAGAASEQ ID NO: 147 TGGTCAATCTCAATGCT TCTCTTGAATTCTGGTCAA TTTTTC TCTCAATGCASEQ ID NO: 148 SEQ ID NO: 149 qRT-PCR Primers Gene Forward ReversehGadd45β CTCCTTAATGTCACGCACGAT GTCCGTGTGAGGGTTCTGTA SEQ ID NO: 150SEQ ID NO: 151 hATCB CATGTACGTTGCTATCCAGGC CTCCTTAATGTCACGCACGATSEQ ID NO: 152 SEQ ID NO: 153

TABLE VIII Pharmacokinetic parameters Z-DTP2 mDTP3 C₀ (μg/mL) 8.73829.065 T_(max) (hr) NA NA AUC to Last (g-hr/mL) 2.085 6.432 T_(1/2) (hr)2.085 1.262 Vβ (ml) 393.600 75.625 Total CL (mL/hr) 130.762 44.181 TotalCL (mL/min/kg) 78.114 27.131 Last Time point 8.0 6.667 MRTINF (hr) 0.9730.281 Vss (mL) 125.898 12.609

TABLE IX [A] Z-DTP2 Dose level 1 2 3 Desidered steady state plasma 1 510 level (CPss) (μM) Desidered steady state plasma 0.746 3.73 7.46 level(CPss) (mg/L) KO (kel × V × Cp) mg/hr 0.0976 0.4879 0.9758 Note:kel/t_(1/2) = 0.693/2.08 = 0.332327 hr⁻¹

TABLE IX [B] mDTP3 Dose level 1 2 3 Desidered steady state plasma 1 5 10level (CPss) (μM) Desidered steady state plasma 0.525 2.625 5.25 level(CPss) (mg/L) KO (kel × V × Cp) mg/hr 0.022 0.109 0.218 Note:kel/t_(1/2) = 0.693/2.08 = 0.332327 hr⁻¹

The invention claimed is:
 1. A compound according to Formula I

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising the compound of claim 1 and a pharmaceuticallyacceptable excipient.